Curcumin analogs with anti-tumor and anti-angiogenic properties

ABSTRACT

The present invention is directed to curcumin analogs exhibiting anti-tumor and anti-angiogenic properties, pharmaceutical formulations including such compounds and methods of using such compounds.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation of U.S. application Ser. No.10/690,462, filed 5 Oct. 21, 2003, now U.S. Pat. No. 7,371,766, which isa continuation of U.S. application Ser. No. 09/729,662, filed Dec. 4,2000, now U.S. Pat. No. 6,664,272, which claims the benefit of U.S.Provisional No. 60/168,913, filed Dec. 3, 1999, all of which areincorporated by reference herein in its entirety.

FIELD OF THE INVENTION

The invention relates to compounds useful for the treatment of cancer,and in particular to compounds exhibiting anti-tumor and anti-angiogenicproperties and methods for using such compounds.

BACKGROUND OF THE INVENTION

Tissue factor (TF) is a sedimentable, integral membrane receptor proteinwith an estimated molecular weight of 42-47 kDa. Peritumor fibrindeposition, which is characteristic of most types of human cancer, isthe result of the local expression of potent procoagulants like tissuefactor (TF) in tumor cells, tumor-associated macrophages (TAMs) andtumor-associated vascular endothelial cells (VECs). In addition to theimportance of TF expression in the pathogenesis of the thromboticcomplications common to cancer patients, increasing evidence links TFexpression to the regulation of tumor angiogenesis, growth andmetastasis. For example, angiogenesis in vivo is inhibited by TFantisense. Further, murine tumor cells transfected to overexpress TFenhance vascular permeability factor (VEGF) transcription andtranslation. Conversely, tumor cells transfected with TF antisensereduce VEGF transcription and translation. VEGF acts specifically onVECs to promote vascular permeability, endothelial cell growth andangiogenesis, and has been shown to induce expression of TF activity inVECs and monocytes and is chemotactic for monocytes, osteoblasts andVECs. Expression of TF and VEGF in cancer cells is further enhancedunder hypoxic condition. Thus, there is evidence to suggest that TF is akey molecule participating in the regulation of VEGF synthesis and,hence, tumor angiogenesis in cancer.

Relatively few compounds exhibiting anti-angiogenic properties useful inthe treatment of cancer have been investigated. Curcumin(diferuloylmethane), the aromatic yellow pigment in curry, turmeric andmustard, is known to have anti-angiogenic, anti-tumor, and anti-tumorpromoting properties. In addition, curcumin exhibits numerous othertherapeutic effects, including anti-oxidative, anti-thrombotic,anti-inflammatory, anti-cholesterol and anti-diabetic properties. Twoother compounds that have received considerable attention are genistein,a soybean-derived isoflavone tyrosine kinase C inhibitor, and linomide,a quinoline-3-carboxaminde. Certain flavonoids, such as apigenin, havebeen shown to be more potent inhibitors of cell proliferation and invitro angiogenesis than genistein. There remains a need in the art forcompounds that exhibit anti-tumor and anti-angiogenic properties for usein cancer therapy.

SUMMARY OF THE INVENTION

The present invention provides a group of curcumin analogs that inhibitTF expression and VEGF expression in cancer cells and in vascularendothelial cells in the tumor microenvironment, thereby blocking tumorangiogenesis and growth, without exhibiting a high level of toxicitywith regard to normal vascular endothelial cells. The anti-angiogenicand anti-tumor compounds of the present invention can be useful in thetreatment of any condition benefiting from angiogenesis inhibition, suchas cancer.

In one aspect, the present invention provides compounds of Formula (I)below.

In another aspect, the present invention provides a pharmaceuticalformulation comprising a compound of Formula (I) or Formula (II) belowin a pharmaceutically acceptable carrier.

In a third aspect, the present invention provides a method of treatingcancerous tissue in a subject, comprising administering an effectiveamount of a compound of Formula (I) or Formula (II) to the subject.Preferably, the compound is administered in a pharmaceuticallyacceptable carrier. The effective amount is preferably an amountsufficient to inhibit VEGF or TF production in the cancerous tissue.

BRIEF DESCRIPTION OF THE DRAWINGS

Having thus described the invention in general terms, reference will nowbe made to the accompanying drawings, wherein:

FIGS. 1A and 1B graphically illustrate the relationship between cellviability and VEGF production of human melanoma cells after treatmentwith the compounds of the present invention at two concentrations;

FIGS. 2A and 2B graphically illustrate the effect of known compounds andthe compounds of the present invention on human melanoma cell viability;

FIGS. 3A and 3B graphically illustrate the effect of known compounds andthe compounds of the present invention on human breast cancer cellviability;

FIGS. 4A and 4B graphically illustrate the effect of known compounds andthe compounds of the present invention on transformed murine endothelialcell viability; and

FIGS. 5A and 5B graphically illustrate the effect of known compounds andthe compounds of the present invention on human endothelial cellviability.

DETAILED DESCRIPTION OF THE INVENTION

The present invention will be described more fully hereinafter,including preferred embodiments thereof. This invention may, however, beembodied in many different forms and should not be construed as limitedto the embodiments set forth herein; rather, these embodiments areprovided so that this disclosure will be thorough and complete, and willfully convey the scope of the invention to those skilled in the art.

As used herein, the term “compound” is intended to refer to a chemicalentity, whether in the solid, liquid or gaseous phase, and whether in acrude mixture or purified and isolated. The terms “alkyl,” “alkene,” and“alkoxy” include straight chain and branched alkyl, alkene, and alkoxy,respectively. The term “lower alkyl” refers to C1-C4 alkyl. The term“alkoxy” refers to oxygen substituted alkyl, for example, of theformulas —OR or —ROR¹, wherein R and R¹ are each independently selectedalkyl. The terms “substituted alkyl” and “substituted alkene” refer toalkyl and alkene, respectively, substituted with one or morenon-interfering substituents, such as but not limited to, C3-C6cycloalkyl, e.g., cyclopropyl, cyclobutyl, and the like; acetylene;cyano; alkoxy, e.g., methoxy, ethoxy, and the like; lower alkanoyloxy,e.g., acetoxy; hydroxy; carboxyl; amino; lower alkylamino, e.g.,methylamino; ketone; halo, e.g. chloro or bromo; phenyl; substitutedphenyl, and the like. The term “halogen” includes fluorine, chlorine,iodine and bromine.

“Aryl” means one or more aromatic rings, each of 5 or 6 carbon atoms.Multiple aryl rings may be fused, as in naphthyl or unfused, as inbiphenyl. Aryl rings may also be fused or unfused with one or morecyclic hydrocarbon, heteroaryl, or heterocyclic rings.

“Substituted aryl” is aryl having one or more non-interfering groups assubstituents.

“Heteroaryl” is an aryl group containing from one to four N, O, or Satoms(s) or a combination thereof, which heteroaryl group is optionallysubstituted at carbon or nitrogen atom(s) with C1-6 alkyl, —CF₃, phenyl,benzyl, or thienyl, or a carbon atom in the heteroaryl group togetherwith an oxygen atom form a carbonyl group, or which heteroaryl group isoptionally fused with a phenyl ring. Heteroaryl rings may also be fusedwith one or more cyclic hydrocarbon, heterocyclic, aryl, or heteroarylrings. Heteroaryl includes, but is not limited to, 5-memberedheteroaryls having one hetero atom (e.g., thiophenes, pyrroles, furans);5 membered heteroaryls having two heteroatoms in 1, 2 or 1,3 positions(e.g., oxazoles, pyrazoles, imidazoles, thiazoles, purines); 5-memberedheteroaryls having three heteroatoms (e.g., triazoles, thiadiazoles);5-membered heteroaryls having 3 heteroatoms; 6-membered heteroaryls withone heteroatom (e.g., pyridine, quinoline, isoquinoline, phenanthrine,5,6-cycloheptenopyridine); 6-membered heteroaryls with two heteroatoms(e.g., pyridazines, cinnolines, phthalazines, pyrazines, pyrimidines,quinazolines); 6-membered heretoaryls with three heteroatoms (e.g.,1,3,5-triazine); and 6-membered heteroaryls with four heteroatoms.

“Substituted heteroaryl” is heteroaryl having one or morenon-interfering groups as substituents.

“Heterocycle” or “heterocyclic” means one or more rings of 5, 6 or 7atoms with or without unsaturation or aromatic character and at leastone ring atom which is not carbon. Preferred heteroatoms include sulfur,oxygen, and nitrogen. Multiple rings may be fused, as in quinoline orbenzofuran.

“Substituted heterocycle” is heterocycle having one or more side chainsformed from non-interfering substituents.

“Non-interfering substituents” are those groups that yield stablecompounds. Suitable non-interfering substituents or radicals include,but are not limited to, halo, C₁-C₁₀ alkyl, C₂-C₁₀ alkenyl, C₂-C₁₀alkynyl, C₁-C₁₀ alkoxy, C₇-C₁₂ aralkyl, C₇-C₁₂ alkaryl, C₃-C₁₀cycloalkyl, C₃-C₁₀ cycloalkenyl, phenyl, substituted phenyl, toluoyl,xylenyl, biphenyl, C₂-C₁₂ alkoxyalkyl, C₇-C₁₂ alkoxyaryl, C₇-C₁₂aryloxyalkyl, C₆-C₁₂ oxyaryl, C₁-C₆ alkylsulfinyl, C₁-C₁₀ alkylsulfonyl,—(CH₂)_(m)—O—(C₁-C₁₀ alkyl) wherein m is from 1 to 8, aryl, substitutedaryl, substituted alkoxy, fluoroalkyl, heterocyclic radical, substitutedheterocyclic radical, nitroalkyl, —NO₂, —CN, —NRC(O)—(C₁-C₁₀ alkyl),—C(O)—(C₁-C₁₀ alkyl), C₂-C₁₀ thioalkyl, —C(O)O—(C₁-C₁₀ alkyl), —OH,—SO₂, ═S, —COOH, —NR₂, carbonyl, —C(O)—(C₁-C₁₀ alkyl)-CF₃, —C(O)—CF₃,—C(O)NR₂, —(C₁-C₁₀ alkyl)-S—(C₆-C₁₂ aryl), —C(O)—(C₆-C₁₂ aryl),—(CH₂)_(m)—O—(CH₂)_(m)—O—(C₁-C₁₀ alkyl) wherein each m is from 1 to 8,—C(O)NR₂, —C(S)NR₂, —SO₂NR₂, —NRC(O)NR₂, —NRC(S)NR₂, salts thereof, andthe like. Each R as used herein is H, alkyl or substituted alkyl, arylor substituted aryl, aralkyl, or alkaryl.

The present invention provides compounds of Formula (I)

-   -   wherein:        -   Y is OH, halogen, or CF₃;        -   Z is H, OH, OR₁, halogen, or CF₃;        -   X₁ and X₂ are independently C or N; and        -   A is selected from the group consisting of:

-   -   wherein n is 1-8; X₃ is O, S, SO, SO₂, NH, or NR₁; Q is NH or        NR₁; and V₁₋₄ are each independently OH, OR₂, or halogen; R₁ and        R₂ are independently H, alkyl, substituted alkyl, aryl,        substituted aryl, heteroaryl, substituted heteroaryl,        heterocycle, substituted heterocycle, acyl, alkoxycarbonyl,        aminocarbonyl, alkylaminocarbonyl or dialkylaminocarbonyl; the        dashed lines indicate the presence of optional double bonds; and        L is the point of bonding of A to the compound structure, with        the proviso that Z is not H when Y is OH, Cl or Br and A is

and pharmaceutically acceptable salts thereof.

The present invention also provides a method of treating canceroustissue in a subject, such as a human or other mammal, comprisingadministering to the subject an effective amount of a compound ofFormula (I) above or a compound of Formula (II)

wherein:

-   -   X₄ is (CH₂)_(m), O, S, SO, SO₂, or NR₁₂, where R₁₂ is H, alkyl,        substituted alkyl, acyl, alkoxycarbonyl, aminocarbonyl,        alkylaminocarbonyl or dialkylaminocarbonyl;    -   m is 1-7;    -   each X₅ is independently N or C—R₁₁;    -   and each R₃-R₁₁ are independently H, halogen, hydroxyl, alkoxy,        CF₃, alkyl, substituted alkyl, alkenyl, alkynyl, cycloalkyl,        substituted cycloalkyl, aryl, substituted aryl, alkaryl,        arylalkyl, heteroaryl, substituted heteroaryl, heterocycle,        substituted heterocycle, amino, alkylamino, dialkylamino,        carboxylic acid, carboxylic ester, carboxamide, nitro, cyano,        azide, alkylcarbonyl, acyl, or trialkylammonium; and    -   the dashed lines indicate optional double bonds;

with the proviso that when X₄ is (CH₂)_(m), m is 2-6, and each X₅ isC—R₁₁, R₃-R₁₁ are not alkoxy, and when X₄ is NR₁₂ and each X₅ is N,R₃-R₁₀ are not alkoxy, alkyl, substituted alkyl, alkenyl, alkynyl,cycloalkyl, substituted cycloalkyl, aryl, substituted aryl, alkaryl,arylalkyl, heteroaryl, substituted heteroaryl, amino, alkylamino,dialkylamino, carboxylic acid, or alkylcarbonyl.

The present invention includes all stereoisomeric configurations of thecompounds of Formula (I) and Formula (II), including both opticalisomers, such as enantiomers and diastereoisomers, and geometric(cis-trans) isomers.

Examples of the compounds of the present invention include, but are notlimited to:

and the like and pharmaceutically acceptable salts thereof. Additionalexemplary compounds are given in the appended examples.

The compounds of the present invention may be prepared according tomethods known in the art, particularly in light of the disclosure andexamples set forth herein. The starting materials used to synthesize thecompounds of the present invention are commercially available or capableof preparation using methods known in the art. For example, somecompounds of the present invention may be prepared by reaction of anaromatic aldehyde, such as hydroxybenzaldehyde or fluoro-substitutedbenzaldehyde, with a ketone, such as acetone, cyclohexanone,cyclopentanone, tetrahydro-4-H-pyran-4-one, N-methyl-4-piperidone,piperidin-4-one, and the like, under basic aldol condensationconditions. Similarly, other compounds of the present invention may beprepared by reaction of an alkoxy-substituted benzaldehyde oranisaldehyde with a ketone. As would be understood, the actual ketone oraldehyde utilized will depend on the type and position of thesubstituents of the desired final compound. The salts of the presentinvention may be prepared, in general, by reaction of a compound of theinvention with the desired acid or base in solution. After the reactionis complete, the salts can be crystallized from solution by the additionof an appropriate amount of solvent in which the salt is insoluble.

The compounds of Formula (I) or Formula (II) can have pharmaceuticalactivity and can be useful in the treatment of a subject suffering fromone or more conditions that would benefit from inhibition ofangiogenesis. For example, the compounds of the present invention can beused in the treatment of cancerous tissue and the tumors associatedtherewith, including breast, colon, prostate and skin cancer. Inaddition, the compounds of the present invention can be useful formediating inflammation, rheumatoid arthritis and certain forms ofdiabetes. Subjects which can be treated include animal subjects,typically vertebrates, including both mammalian (e.g., human, cat, dog,cow, horse, sheep, pig, monkey, ape, etc.) and avian subjects (e.g.,chicken, turkey, duck, goose, quail, pheasant, etc.). It is believed,for example, that administering an effective amount of a compound ofFormula (I) or Formula (II) to a subject can result in inhibition ofangiogenesis in cancerous tissue. Thus, the present invention canprovide methods for treating tumor-bearing subjects in which thecompounds of the invention are administered to the subject in need ofsuch treatment in an amount effective and in a manner effective tocombat such tumors, for example, by virtue of inhibition of angiogenesiswithin the tumor. The anti-angiogenesis effect is believed to result, atleast in part, from inhibition of TF and/or VEGF production in thetumor. In addition, it is believed that the compounds of the presentinvention can be used as a prophylactic treatment to prevent certaintypes of inflammatory skin conditions including, but not limited to,dermatitis and mild cases of skin cancer.

The compounds of Formula (I) or Formula (II) may be administered per seor in the form of a pharmaceutically acceptable salt. When used inmedicine, the salts of the compounds of Formula (I) or Formula (II)should be both pharmacologically and pharmaceutically acceptable, butnon-pharmaceutically acceptable salts may conveniently be used toprepare the free active compound or pharmaceutically acceptable saltsthereof and are not excluded from the scope of this invention. Suchpharmacologically and pharmaceutically acceptable salts can be preparedby reaction of a compound of Formula (I) or Formula (II) with an organicor inorganic acid, using standard methods detailed in the literature.Examples of useful salts include, but are not limited to, those preparedfrom the following acids: hydrochloric, hydrobromic, sulfuric, nitric,phosphoric, maleic, acetic, salicyclic, p-toluenesulfonic, tartaric,citric, methanesulphonic, formic, malonic, succinic,naphthalene-2-sulphonic and benzenesulphonic, and the like. Also,pharmaceutically acceptable salts can be prepared as alkaline metal oralkaline earth salts, such as sodium, potassium, or calcium salts of thecarboxylic acid group.

Thus the present invention also provides pharmaceutical formulations orcompositions, both for veterinary and for human medical use, whichcomprise the compounds of Formula (I) or Formula (II) or apharmaceutically acceptable salt thereof with one or morepharmaceutically acceptable carriers thereof and optionally any othertherapeutic ingredients, such as other chemotherapeutic agents. Thecarrier(s) must be pharmaceutically acceptable in the sense of beingcompatible with the other ingredients of the formulation and not undulydeleterious to the recipient thereof.

The compositions includes those suitable for oral, rectal, topical,nasal, ophthalmic, or parenteral (including intraperitoneal,intravenous, subcutaneous, or intramuscular injection) administration.The compositions may conveniently be presented in unit dosage form andmay be prepared by any of the methods well known in the art of pharmacy.All methods include the step of bringing the active agent intoassociation with a carrier that constitutes one or more accessoryingredients. In general, the compositions are prepared by uniformly andintimately bringing the active compound into association with a liquidcarrier, a finely divided solid carrier or both, and then, if necessary,shaping the product into desired formulations.

Compositions of the present invention suitable for oral administrationmay be presented as discrete units such as capsules, cachets, tablets,lozenges, and the like, each containing a predetermined amount of theactive agent as a powder or granules; or a suspension in an aqueousliquor or non-aqueous liquid such as a syrup, an elixir, an emulsion, adraught, and the like.

A tablet may be made by compression or molding, optionally with one ormore accessory ingredients. Compressed tablets may be prepared bycompressing in a suitable machine, with the active compound being in afree-flowing form such as a powder or granules which is optionally mixedwith a binder, disintegrant, lubricant, inert diluent, surface activeagent or dispersing agent. Molded tablets comprised with a suitablecarrier may be made by molding in a suitable machine.

A syrup may be made by adding the active compound to a concentratedaqueous solution of a sugar, for example sucrose, to which may also beadded any accessory ingredient(s). Such accessory ingredients mayinclude flavorings, suitable preservatives, an agent to retardcrystallization of the sugar, and an agent to increase the solubility ofany other ingredient, such as polyhydric alcohol, for example, glycerolor sorbitol.

Formulations suitable for parental administration conveniently comprisea sterile aqueous preparation of the active compound, which can beisotonic with the blood of the recipient.

Nasal spray formulations comprise purified aqueous solutions of theactive agent with preservative agents and isotonic agents. Suchformulations are preferably adjusted to a pH and isotonic statecompatible with the nasal mucous membranes.

Formulations for rectal administration may be presented as a suppositorywith a suitable carrier such as cocoa butter, or hydrogenated fats orhydrogenated fatty carboxylic acids.

Ophthalmic formulations are prepared by a similar method to the nasalspray, except that the pH and isotonic factors are preferably adjustedto match that of the eye.

Topical formulations comprise the active compound dissolved or suspendedin one or more media such as mineral oil, petroleum, polyhydroxyalcohols or other bases used for topical formulations. The addition ofother accessory ingredients as noted above may be desirable.

Further, the present invention provides liposomal formulations of thecompounds of Formula (I) or Formula (II) and salts thereof. Thetechnology for forming liposomal suspensions is well known in the art.When the compound of Formula (I) or Formula (II) or salt thereof is anaqueous-soluble salt, using conventional liposome technology, the samemay be incorporated into lipid vesicles. In such an instance, due to thewater solubility of the compound or salt, the compound or salt will besubstantially entrained within the hydrophilic center or core of theliposomes. The lipid layer employed may be of any conventionalcomposition and may either contain cholesterol or may becholesterol-free. When the compound or salt of interest iswater-insoluble, again employing conventional liposome formationtechnology, the salt may be substantially entrained within thehydrophobic lipid bilayer that forms the structure of the liposome. Ineither instance, the liposomes that are produced may be reduced in size,as through the use of standard sonication and homogenization techniques.The liposomal formulations containing the compounds of Formula (I) orFormula (II) or salts thereof, may be lyophilized to produce alyophilizate which may be reconstituted with a pharmaceuticallyacceptable carrier, such as water, to regenerate a liposomal suspension.

Pharmaceutical formulations are also provided which are suitable foradministration as an aerosol, by inhalation. These formulations comprisea solution or suspension of the desired compound of Formula (I) orFormula (II) or a salt thereof or a plurality of solid particles of thecompound or salt. The desired formulation may be placed in a smallchamber and nebulized. Nebulization may be accomplished by compressedair or by ultrasonic energy to form a plurality of liquid droplets orsolid particles comprising the compounds or salts.

In addition to the aforementioned ingredients, the compositions of theinvention may further include one or more accessory ingredient(s)selected from the group consisting of diluents, buffers, flavoringagents, binders, disintegrants, surface active agents, thickeners,lubricants, preservatives (including antioxidants) and the like.

Preferably, for purposes of cancer therapy, a compound of Formula (I) orFormula (II) is administered to the subject in an amount sufficient toinhibit production of TF or VEGF, thereby inhibiting angiogenesis.However, the therapeutically effective dosage of any specific compoundwill vary somewhat from compound to compound, patient to patient, andwill depend upon the condition of the patient and the route of delivery.As a general proposition, a dosage from about 0.5 to about 20 mg/kg bodyweight, preferably from about 1.0 to about 5.0 mg/kg, will havetherapeutic efficacy. When administered conjointly with otherpharmaceutically active agents, even less of the compounds of Formula(I) or Formula (II) may be therapeutically effective. The compound ofFormula (I) or Formula (II) may be administered once or several times aday. The duration of the treatment may be once per day for a period offrom two to three weeks and may continue for a period of months or evenyears. The daily dose can be administered either by a single dose in theform of an individual dosage unit or several smaller dosage units or bymultiple administration of subdivided dosages at certain intervals.

For the following examples, RPMI-7951 human melanoma, MDA-MB-231 andMDA-MB-435 human breast cancer cell lines were purchased from AmericanType Cell Collection (Rockville, Md.). HUVECs were obtained from theDepartment of Dermatology, Emory University. Murine endothelial cellsinfected with simian virus 40 (SV40) large T antigen and activated H-ras(SVR), were a kind gift from Dr. Jack Arbiser at Emory. RPMI-7951,MDA-MB-231 and MDA-MB-435 cell lines were cultured in MEM-alpha medium(GIBCO-BRL, Long Island, N.Y.) containing 10% fetal bovine serum(RPMI-7951, MDA-MB-231) or 5% FBS (MDA-MB-435) at 37° C. and under 5%CO₂/95% air. SVR cells were cultured in DMEM (Mediatech cellgro)containing 10% FBS and 2 mM L-glutamine. Complete HUVEC media was a giftfrom the Cell Culture Center in the Department of Dermatology, EmoryUniversity. The cells were cultured in 48 well plates in all of theexperiments described.

Neutral Red Assay was utilized to determine the effect of the compoundsof the present invention on cell viability. Neutral Red was purchasedfrom GIBCO-BRL (Long Island, N.Y.). Cells were plated at a concentrationof 20,000 cells/well and cultured overnight. Compounds or vehicle (DMSO0.1%) were then added and the plates were incubated for 72 hours.Supernatant from each well was either aspirated or collected and mediacontaining Neutral Red (GIBCO-BRL, Long Island, N.Y.) at a concentrationof 15 μl/ml was then added to each well. The plates were then incubatedat 37° C. for 30 minutes. Next, the cells were washed with twice withPBS and alcoholic-HCl (0.5N—HCl/35% ethanol) was added to each well. Theplates were then placed on a plate shaker until all residues weresolubilized (pink color). The solubilized mixtures were then transferredto a 96 well plate and the absorbances were read on a micro-test platereader at a wavelength of 570 nM.

A VEGF enzyme-linked immunosorbent assay (ELISA) was utilized todetermine the effect of the compounds of the present invention on VEGFproduction of a variety of human cancer cell lines. For the VEGF assay,cells were plated at a concentration of 80,000 cells/well and culturedovernight. Compounds or vehicle (DMSO 0.1%) were then added and theplates were incubated for 72 hours. Supernatant was then collected fromeach well and frozen in a −80° C. freezer until needed. Cell viabilitywas determined by Neutral Red Assay. VEGF ELISA Kit (R & D, Minneapolis,Minn.) was used to determine the amount of VEGF in the culturesupernatants. The ELISA was carried out according to the manufacturer'sprocedure.

A TF ELISA assay was utilized to determine the effect of the compoundsof the present invention on TF production of human cancer cell lines.For the TF assay, cells were plated at a concentration of 80,000cells/well and cultured overnight. Compounds or vehicle (DMSO 0.1%) werethen added and the plates were incubated for 72 hours. Cells weretreated with 1% Triton X-100 in PBS and left overnight at 4° C.overnight to solubilize TF. Supernatant was then collected from eachwell and frozen until needed. IMUBIND Tissue Factor ELISA Kit (AmericanDiagnostica Inc, Greenwich, Conn.) was used to determine TFconcentration in each sample. The ELISA was carried out according tomanufacturer's procedure.

EXPERIMENTAL Example 1 Preparation of EF1, EF2, EF3, EF4, EF25, EF31,EF34 Compounds

The compounds of this series were all synthesized by the followingprocedure: Aqueous NaOH (20 wt %, 15 ml, 75 mmol) was added dropwise toa vigorously stirred solution of hydroxybenzaldehyde (51 mmol) andketone (25 mmol) in EtOH abs (20 mL). The reaction was stirred at roomtemperature for 48 hrs, H₂O dist (100 mL) was added, and the purplesolution was neutralized by gently bubbling CO₂ through it. Theprecipitating yellow solid was filtered off, washed with H₂O dist anddried under vacuum. The products were purified by recrystallization. Adescription of each compound obtained by the above process is givenbelow.

1,5-Bis(4-hydroxyphenyl)penta-1,4-dien-3-one (EF1): yellow solid (6%),mp 236° C. (acetone/H₂O). ¹H NMR (400 MHz, CD₃OD) δ 7.71 (2H, d, J=16Hz), 7.58 (4H, d, J=8.8 Hz), 7.07 (2H, d, J=16 Hz), 6.84 (4H, d, J=8.4Hz). ¹³C NMR (100 MHz, CD₃OD) δ 191.9, 161.8, 145.3, 131.8, 127.8,123.6, 117.1. EIHRMS: m/z 266.0943 (M⁺, C₁₇H₁₄O₃ requires 266.0943).

1,5-Bis(2-hydroxyphenyl)penta-1,4-dien-3-one (EF2): yellow solid (75%),mp 155° C. (acetone/H₂O). ¹H NMR (400 MHz, CD₃OD) δ 8.09 (2H, d, J=16Hz), 7.63 (2H, dd, J=8.4 Hz, J=1.6 Hz), 7.31 (2H, d, J=16 Hz), 7.24 (2H,td, J=7.6 Hz, J=1.6 Hz), 6.88 (t, 4H, J=7.2 Hz). ¹³C NMR (100 MHz,CD₃OD) δ 192.8, 158.9, 141.0, 133.1, 130.2, 126.3, 123.2, 121.0, 117.2.Anal. Calcd for C₁₇H₁₄O₃: C, 78.68; H, 5.30. Found: C, 76.56; H, 5.32.EIHRMS: m/z 248.0837 ((M-H₂O)⁺, C₁₇H₁₂O₂ requires 248.0837).

1,5-Bis(3-hydroxyphenyl)penta-1,4-dien-3-one (EF3): yellow solid (15%),mp 198-200° C. (acetone/H₂O). ¹H NMR (400 MHz, CD₃OD) δ 7.70 (2H, d,J=16 Hz), 7.24 (2H, t, J=7.6 Hz), 7.17 (2H, d, J=16 Hz), 7.17 (2H, d,J=8 Hz), 7.11 (2H, s), 6.73 (2H, dd, J=8 Hz, J=2.4 Hz). ¹³C NMR (100MHz, CD₃OD) δ 191.6, 159.3, 145.5, 137.6, 131.2, 126.4, 121.4, 119.1,115.9. Anal. Calcd for C₁₇H₁₄O₃: C, 78.68; H, 5.30. Found: C, 76.41; H,5.48. EIHRMS: m/z 266.0943 (M⁺, C₁₇H₁₄O₃ requires 266.0943).

2,6-Bis(2-hydroxybenzylidene)cyclohexanone (EF4): yellow solid (70%,recrystallized from acetone/H₂O). ¹H NMR (400 MHz, CD₃OD) δ 7.98 (2H,s), 7.32 (2H, dd, J=7.6 Hz, J=1.2 Hz), 7.19 (2H, td, J=7.6 Hz, J=1.2Hz), 6.86 (4H, m), 2.86 (4H, m), 1.75 (2H, m). ¹³C (100 MHz, CD₃OD) δ192.8, 158.2, 137.3, 134.5, 131.5, 124.5, 120.2, 116.6, 29.9, 24.8.Anal. Calcd for C₂₀H₁₈O₃: C, 78.41; H, 5.92. Found: C, 78.15; H, 6.03.EIHRMS: m/z 306.1263 (M⁺, C₂₀H₁₈O₃ requires 306.1256).

3,5-Bis(2-hyrdoxybenzylidene)tetrahydro-4-H-pyran-4-one (EF25): yellowsolid (60%, recrystallized from acetone/H₂O). ¹H NMR (400 MHz, CD₃OD) δ8.08 (2H, s), 7.24 (2H, td, J=8.4 Hz, J=1.6 Hz), 7.09 (2H, dd, J=7.6 Hz,J=1.6 Hz), 7.90-7.86 (4H, m), 4.84 (4H, d, J=1.6 Hz). ¹³C NMR (100 MHz,CD₃OD) δ 187.8, 158.5, 133.9, 132.5, 131.9, 123.3, 120.5, 116.8, 70.0.Anal. Calcd for C₁₉H₁₆O₄: C, 74.01; H, 5.23. Found: C, 73.23; H, 5.23.EIHRMS: m/z 290.0933 (M⁺, C₁₉H₁₆O₄ requires 290.0943).

2,5-Bis(2-hydroxyphenyl)cyclopentanone (EF31): yellow solid (81%,recrystallized from acetone). ¹H NMR (400 MHz, CD₃OD) δ 8.00 (2H, s),7.57 (2H, dd, J=8.0 Hz, J=1.2 Hz), 7.22 (2H, td, J=8.0 Hz, J=1.6 Hz),6.88 (4H, m), 3.06 (4H, s).

3,5-Bis(2-hydroxybenzylidene)1-methyl-4-piperidone (EF34): yellow solid(75%, recrystallized from methanol/H₂O). ¹H NMR (400 MHz, CD₃OD) δ 8.11(2H, s), 7.23 (4H, t, J=7.6 Hz), 6.88 (4H, t, J=8.0 Hz), 3.76 (4H, d,J=1.2 Hz), 2.42 (3H, s). ¹³C NMR (100 MHz, CD₃OD) δ 188.40, 158.58,135.09, 133.09, 132.27, 131.64, 123.59, 120.38, 116.87. HREIMS: m/z303.1259 (M⁺-H₂O, C₂₀H₁₇NO₂ requires 303.1259).

Example 2 Preparation of EF8, EF9, EF10, EF23, EF29, EF30, EF33

The compounds of this series were all synthesized by the followingprocedure: A solution of fluoro-substituted benzaldehyde (5.00 mmol) inethanol abs. (1 mL) was added at room temperature over a period of 5min, with stirring, to a solution of NaOH (0.75 mmol) and ketone(acetone, tetrahydro-4-H-pyran-4-one, N-methyl-piperodin-4-one) (2.50mmol) in a mixture of ethanol abs (7 mL) and H₂O dist. (7 mL). Thesolution turns yellow immediately, and usually a yellow precipitatestarts forming within 10 min (except EF8, where an oil forms). Thereaction was stirred at room temperature for 3 hrs., the yellow solidfiltered off, washed with water and hexanes and dried under vacuum. Theproduct was obtained in analytically pure form, further purification wasonly necessary where indicated. A description of each compound obtainedby the above process is given below.

1,5-Bis(2-fluorophenyl)penta-1,4-dien-3-one (EF8): yellow solid (50%).¹H NMR (400 MHz, CDCl₃) δ 7.86 (2H, d, J=16 Hz), 7.63 (2H, td, J=7.6 Hz,J=1.6 Hz), 7.42-7.35 (2H, m), 7.18 (2H, d, J=16 Hz), 7.26-7.10 (4H, m).¹³C NMR (100 MHz, CDCl₃) δ 189.2, 161.9 (d, J=253.4 Hz), 136.3 (d, J=2.3Hz), 132.1 (d, J=9.1 Hz), 129.5 (d, J=2.2 Hz), 127.8 (d, J=6.1 Hz),124.7 (d, J=3.8 Hz), 123.1 (d, J=11.4 Hz), 116.4 (d, J=22.0 Hz). Anal.Calcd for C₁₇H₁₂F₂O: C, 75.55; H, 4.48; F, 14.06. Found: C, 75.30; H,4.55. EIHRMS: m/z 270.0865 (M⁺, C₁₇H₁₂F₂O requires 270.0856).

1,5-Bis(2,4-difluorophenyl)penta-1,4-dien-3-one (EF9): yellow solid(72%). ¹H NMR (300 MHz, CDCl₃) δ 7.79 (2H, d, J=16.2 Hz), 7.62 (2H, dd,J=15 Hz, J=8.4 Hz), 7.10 (2H, d, J=16 Hz), 8.85-8.97 (4H, m). ¹³C NMR(75 MHz, CDCl₃) δ 188.5, 164.8 (dd, J=169.6 Hz, J=12.2 Hz), 161.3 (dd,J=160.3 Hz, J=12.2 Hz), 135.3, 130.7 (q, J=10.0 Hz, J=4.6 Hz), 127.3,119.5, 112.3 (d, J=21.9 Hz), 104.9 (t, J=25.4 Hz). Anal. Calcd forC₁₇H₁₀F₄O: C, 66.67; H, 3.29. Found: C, 66.38; H, 3.41. EIHRMS: m/z306.0654 (M⁺, C₁₇H₁₀OF₄ requires 306.0668).

1,5-Bis(3,4-difluorophenyl)penta-1,4-dien-3-one (EF10): yellow solid(86%). ¹H NMR (400 MHz, CDCl₃) δ 7.64 (2H, d, J=16 Hz), 7.44 (2H, ddd,J=9.6 Hz, 7.6 Hz, J=2 Hz), 7.32-7.36 (2H, m), 7.18-7.25 (2H, m), 6.96(2H, d, J=16 Hz). ¹³C NMR (100 MHz, CDCl₃) δ 180.0, 152.7 (dd, J=105.4Hz, J=12.9 Hz), 150.2 (dd, J=101.7 Hz, J=12.9 Hz), 141.5, 132.1 (t,J=5.2 Hz), 126.2, 125.5 (q, J=6 Hz, J=3 Hz), 118.2 (d, J=17.5 Hz), 116.7(d, J=20.5 Hz). Anal. Calcd for C₁₇H₁₀F₄O: C, 66.67; H, 3.29. Found: C,66.54; H, 3.28. EIHRMS: m/z 306.0671 (M⁺, C₁₇H₁₀OF₄ requires 306.0668).

1,5-Bis(2,6-difluorophenyl)penta-1,4-dien-3-one (EF23): yellow solid(91%). ¹H NMR (400 MHz, CDCl₃) δ 7.82 (2H, d, J=16.4 Hz), 7.35 (2H, d,J=16 Hz), 7.33 (2H, td, J=8 Hz, J=2.4 Hz), 6.96 (4H, t, J=8.8 Hz). ¹³CNMR (100 MHz, CDCl₃) δ 189.5, 162.2 (dd, J=1017.6 Hz, J=29.2 Hz), 131.6(t, J=10.9 Hz), 131.0 (t, J=8.7 Hz), 129.9, 113.0 (t), 112.1 (d, J=25.6Hz). Anal. Calcd for C₁₇H₁₀FO₄: C, 66.67; H, 3.29. Found: C, 66.46; H,3.26, EIHRMS: m/z 306.0657 (M⁺, C₁₇H₁₀FO₄ requires 306.0668).

3,5-Bis(2-fluorobenzylidene)tetrahydro-4-H-pyran-4-one (EF29): (84%,recrystallized from hot ethanol). ¹H NMR (400 MHz, CDDl₃) δ 7.92 (2H, d,J=1.2 Hz), 7.40-7.36 (2H, m), 7.20-7.18 (4H, m), 7.13 (2H, t, J=9.6 Hz),4.80 (s, 4H, CH₂). ¹³C NMR (100 MHz, CDCl₃) δ 185.1, 161.0 (d, J=250.7Hz), 134.8, 131.5 (d, J=8.7 Hz), 131.1 (d, J=2.2 Hz), 129.6 (d, J=3.7Hz), 124.3 (d, J=3.7 Hz), 122.8 (d, J=13.8 Hz), 116.2 (d, J=21.9 Hz),68.9 (d, J=5.1 Hz). Anal. Calcd for C₁₉H₁₄F₂O₂: C, 73.07; H, 4.52.Found: C, 73.07; H, 4.47. EIHRMS; m/z 312.0950 (M⁺, C₁₉H₁₄F₂O₂ requires312.0962).

3,5-Bis(2,4-difluorobenzylidene)tetrahydro-4-H-pyran-4-one (EF30): (82%,recryst. from ethanol/H₂O). ¹H NMR (400 MHz, CDCl₃) δ 7.85 (2H, s),7.21-7.14 (1H, m), 6.98-6.87 (2H, m), 4.77 (4H, s). 13C NMR (100 MHz,CDCl₃) δ 184.81, 163.95 (dd, J=240 Hz, J=12 Hz), 161.43 (dd, J=265 Hz,J=12 Hz), 134.48, 132.02 (dd, J=9.5 Hz, J=4.3 Hz), 128.62, 119.19,112.00 (dd, J=3.7 Hz, J=21.9 Hz), 104.85 (t, J=25.5 Hz), 68.81 (d, J=4.4Hz). Anal. Calcd for C₁₉H₁₂F₄O₂; C, 65.52; H, 3.47. Found: C, 65.67; H,3.43. EIHRMS: m/z 348.0761 (M⁺, C₁₉H₁₄F₂O₂ requires 348.0773).

3,5-Bis(2-fluorobenzylidene)l-methyl-4-piperidone (EF33): (82%, yellowsolid). ¹H NMR (400 MHz, CDCl₃) δ 7.90 (2H, s), 7.40-7.33 (2H, m), 7.28(2H, td, J=7.6 Hz, J=1.2 Hz), 7.18 (2H, td, J=7.6 Hz, J=0.8 Hz), 7.12(2H, td, J=10.0 Hz, J=0.8 Hz), 3.64 (4H, s), 2.40 (3H, s). ¹³C NMR (100MHz, CDCl₃) δ 186.3, 161.1 (d, J=251 Hz), 134.9, 131.1 (d, J=8.1 Hz),131.0 (d, J=3.0 Hz), 129.7 (d, J=3.6 Hz), 124.1 (d, J=2.9 Hz), 123.4 (d,J=13.9 Hz), 116.1 (d, J=21.8 Hz), 57.2, 45.8. Anal. Calcd forC₂₀H₁₇F₂ON: C, 73.83; H, 5.27; N, 4.30. Found: C, 73.59; H, 5.32; N,4.39. EIHRMS: m/z 325.1278 (M₊, C₂₀H₁₇F₂ON requires 325.1278).

Example 3 Preparation of EF11, EF12, EF13, EF14, EF15

The compounds of this series were all synthesized by the followingprocedure: Bis-diethylphosphorylmethylsulfide, -sulfoxide and -sulfonewere obtained according to literature procedures (Tetrahedron, 1992, 48,8065-8072; Phosphorus Sulfur 1981, 10, 369-374). A solution ofphosphonate (0.60 mmol) and aldehyde (1.25 mmol) in CH₂Cl₂ (3 mL) wasadded to the heterogeneous mixture of 50% aqueous NaOH (2 mL) and CH₂Cl₂(2 mL), containing triethylbenzylammonium chloride (TEBA, 0.06 mmol).The reaction was stirred at room temperature over night, the product wasextracted from the reaction mixture with CH₂Cl₂ and purified by columnchromatography. A description of each compound obtained by the aboveprocess is given below.

3,5-Bis(2-hydroxybenzylidene)-sulfone (EF11): yellow solid (45%, 30%EtOAc/hexanes). ¹H NMR (400 MHz, CD₃OD) δ 7.77 (2H, d, J=15.6 Hz), 7.48(2H, dd, J=8.4 Hz, J=2.0 Hz), 7.25 (2H, td, J=8.0 Hz, J=2.5 Hz), 7.22(2H, d, J=15.6 Hz), 6.88-6.84 (4H, m), 4.91 (2H, s-br). ¹³C NMR (100MHz, CD₃OD) δ 158.94, 140.21, 133.55, 131.63, 127.84, 121.15, 121.03,117.23.

3,5-Bisbenzylidenesulfone (EF12): white solid (78%, 20% EtOAc/hexanes).¹H NMR (400 MHz, CDCl₃) δ 7.65 (2H, d, J=15.2 Hz, H3), 7.53-7.50 (4H,m), 7.44-7.40 (6H m), 6.86 (2H, d, J=15.6 Hz, H2). ¹³C NMR (100 MHz,CDCl₃) δ 143.7, 132.7, 131.5, 129.3, 128.8, 126.5. Anal. Calcd forC₁₆H₁₄SO₂: C, 71.09; H, 5.22, S, 11.86. Found: C, 70.88; H, 5.21; S,12.01. EIHRMS: m/z 270.0715 (M⁺, C₁₆H₁₄SO₂ requires 270.0731).

E,E-3,5-Bisbenzylidenesulfoxide (EF13): white solid (33%, 20% EtOAc,hexanes). ¹H NMR (300 MHz, CDCl₃) δ 7.52-7.46 (4H, m), 7.42-7.35 (6H,m), 7.31 (2H, d, J=15.3 Hz), 6.87 (2H, d, J=15.3 Hz). ¹³C NMR (75 MHz,CDCl₃) δ 137.0, 133.9, 131.1, 130.0, 129.1, 127.9. Anal. Calcd forC₁₆H₁₄SO: C, 75.56; H, 5.55, S, 12.60. Found: C, 75.33; H, 5.60; S,12.60.

E,Z-3,5-Bisbenzylidenesulfoxide (EF14): white solid (15%, 20% EtOAc,hexanes). ¹H NMR (300 MHz, CDCl₃) δ 7.52-7.40 (10H, m), 7.33 (1H, d,J=15.3 Hz), 7.12 (1H, d, J=10.5 Hz), 6.93 (1H, d, J=15.6 Hz), 6.39 (1H,d, J=10.5 Hz). ¹³C NMR (75 MHz, CDCl₃) δ 138.5, 136.3, 135.5, 134.1,130.1, 129.8, 129.6, 129.0, 128.8, 127.8. Anal. Calcd for C₁₆H₁₄SO: C,75.56; H, 5.55, S, 12.60. Found: C, 75.34; H, 5.54; S, 12.57.

3,5-Bisbenzylidenesulfide (EF15): white solid (20%, 5% EtOAc/hexanes)mixture of E,E and E,Z (ca. 2.5:1). ¹H NMR (400 MHz, CDCl₃) δ 7.40-7.10(20H, m), 6.79 (2H, d, J=15.6 Hz), 6.76 (1H, d, J=15.6 Hz), 6.62 (2H, d,J=15.6 Hz), 6.61 (1H, d, J=15.6 Hz), 6.53 (1H, J=10.8 Hz), 6.41 (1H, d,J=10.8 Hz). Anal. Calcd for C₁₆H₁₄S: C, 80.63; H, 5.92; S, 13.45. Found:C, 80.40; H, 5.93; S, 13.39.

Example 4 Preparation of EF16, EF17, EF18, EF27, EF28

The compounds of this series were all synthesized by the followingprocedure: NaOH (0.10 mmol) was added as a solid to a stirred solutionof methoxy-substituted benzaldehyde/anisaldehyde (2.50 mmol) and ketone(acetone, tetrahydro-4-H-pyranone) in EtOH abs (5 mL). A yellow solidstarted forming within 1 hr. The reaction was stirred at roomtemperature for 20 hrs, the product filtered off, washed with cold EtOHabs and H₂O dist and dried under vacuum. A description of each compoundobtained by the above process is given below.

1,5-Bis(2-methoxphenyl)penta-1,4-dien-3-one (EF16): yellow solid (60%).Mp 123-124° C. (EtOH). ¹H NMR (400 MHz, CDCl₃) δ 8.07 (2H, d, J=16 Hz),7.63 (2H, dd, J=7.6 Hz, J=1.6 Hz), 7.37 (2H, ddd, J=7.2 Hz, J=1.6 Hz,J=1.2 Hz), 7.18 (2H, d, J=16 Hz), 6.99 (2H, t, J=7.6 Hz), 6.93 (2H, d,J=8.4 Hz). ¹³C NMR (100 MHz, CDCl₃) δ 190.2, 158.7, 138.4, 131.8, 128.9,126.4, 124.1, 120.9, 111.4, 55.7. Anal. Calcd for C₁₉H₁₈O₃: C, 77.53; H,6.16. Found: C, 77.26; H, 6.17. EIHRMS: m/z 294.1256 (M⁺, C₁₉H₁₈O₃requires 294.1256).

1,5-Bis(3-methoxyphenyl)penta-1,4-dien-3-one (EF17): yellow solid (40%,chromatography using 20% EtOAc/hexanes). ¹H NMR (300 MHz, CDCl₃) δ 7.70(2H, d, J=15.9 Hz), 7.33 (2H, t, J=7.6 Hz), 7.21 (2H, d, J=7.8 Hz), 7.13(2H, t, J=2.4 Hz), 7.06 (2H, d, J=15.9 Hz), 6.96 (2H, ddd, J=8.1 Hz,J=2.4 Hz, J=0.9 Hz). ¹³C NMR (75 MHz, CDCl₃) δ 188.83, 159.96, 143.31,136.21, 130.03, 125.72, 121.20, 116.46, 113.36, 55.55.

1,5-Bis(4-methoxyphenyl)penta-1,4-dien-3-one (EF18): yellow solid (93%).Mp 129-130° C. (EtOH). ¹H NMR (300 MHz, CDCl₃) δ 7.69 (2H, d, J=15.9 Hz,3), 7.56 (4H, d, J=9 Hz), 6.95 (2H, d, J=15.6 Hz), 6.92 (4H, d, J=8.7Hz). ¹³C NMR (75 MHz, CDCl₃) δ 188.8, 161.5, 142.7, 130.2, 127.7, 126.6,114.5, 55.6 Anal. Calcd for C₁₉H₁₈O₃: C, 77.53; H, 6.16. Found: C,77.31; H, 6.217. EIHRMS: m/z 294.1268 (M⁺, C₁₉H₁₈O₃ requires 294.1256).

3,5-Bis(4-methoxybenzylidene)tetrahydro-4-H-pyran-4-one (EF27): yellowsolid (40%). ¹H NMR (400 MHz, CDCl₃) δ 7.79 (2H, s), 7.29 (4H, d, J=8.8Hz), 6.95 (4H, d, J=9.2 Hz), 4.93 (4H, d, J=1.6 Hz), 3.85 (6H, s). ¹³CNMR (100 MHz, CDCl₃) δ 185.6, 160.8, 136.2, 132.7, 131.4, 127.7, 114.4,68.9, 55.6. Anal. Calcd for C₂₁H₂₀O₄: C, 74.98; H, 5.99. Found: C,74.81; H, 6.01. EIHRMS: m/z 336.1362 (M⁺, C₂₁H₂₀O₄ requires 336.1361).

3,5-Bis(2-methoxybenzylidene)tetrahydro-4-H-pyran-4-one (EF28): yellowsolid (67%). ¹H NMR (400 MHz, CDCl₃) δ 8.09 (2H, s), 7.36 (2H, td, J=8.8Hz, J=1.6 Hz), 7.07 (2H, dd, J=7.6 Hz, J=1.6 Hz), 6.97 (2H, t, J=7.6Hz), 6.93 (2H, d, J=8 Hz), 4.81 (4H, d, J=1.6 Hz), 3.87 (6H, s). ¹³C NMR(100 MHz, CDCl₃) δ 186.0, 158.6, 133.3, 132.5, 131.1, 130.7, 124.1,120.3, 111.0, 69.1, 55.7. Anal. Calcd for C₂₁H₂₀O₄: C, 74.98; H, 5.99.Found: C, 74.84; H, 5.92. EIHRMS: m/z 336.1370) (M⁺, C₂₁H₂₀O₄ requires336.1361).

Example 5 Preparation of EF19, EF20, EF32

The compounds of this series were all synthesized by the followingprocedure: A solution of substituted dienone (0.69 mmol) in EtOH abs (29mL) was subject to hydrogenation at 33 psi using Raney Nickel as thecatalyst for 4 hrs. Filtration through CELITE and concentration undervacuum yielded the crude product, which was purified by chromatographyon silica gel using 25% EtOAc/hexanes. A description of each compoundobtained by the above process is given below.

1,5-Bis(2,4-difluorophenyl)-pentan-3-ol (EF19): white solid (92%). ¹HNMR (300 MHz, CDCl₃) δ 7.17-7.09 (2H, m), 6.82-6.72 (4H, m), 3.60 (1H,m), 2.84-2.60 (4H, m), 1.80-1.75 (4H, m), 1.59 (1H, s-br). ¹³C NMR (75MHz, CDCl₃) δ 162.9 (dd, J=34.4 Hz, J=12 Hz), 159.7 (dd, J=35 Hz, J=12Hz), 131.1 (t, J=9 Hz), 124.6 (dd, J=19.7 Hz, J=3.8 Hz), 111.2 (dd,J=20.5 Hz, J=3.4 Hz), 103.8 (t, J=25.7 Hz), 70.6, 38.0, 25.1. Anal.Calcd for C₁₇H₁₆FO₄: C, 65.38; H, 5.16. Found: C, 65.64; H, 5.24.EIHRMS: m/z 312.1137 (M⁺, C₁₇H₁₆FO₄ requires 312.1137).

1,5-Bis(3,4-difluorophenyl)-pentan-3-ol (EF20): white solid (81%). ¹HNMR (300 MHz, CDCl₃) δ 7.07-6.91 (4H, m), 6.87-6.80 (2H, m), 3.57 (1H,m), 2.77-2.54 (4H, m), 1.73 (4H, t), 1.41 (1H, s-br). ¹³C NMR (75 MHz,CDCl₃) δ 151.2 (dd, J=107.6 Hz, J=12.5 Hz), 148.0 (dd, J=105.8 Hz,J=12.5 Hz), 138.9 (t), 124.3 (d, J=5.8 Hz), 117.3, 117.1, 70.4, 39.3,31.5. EIHRMS: m/z 312.1138 (M⁺, C₁₇H₁₆FO₄ requires 312.1137).

1,5-Bis(2-hydroxyphenyl)-pentan-3-ol (EF32): white solid (45%). ¹H NMR(300 MHz, CDCl₃) δ 7.56 (2H, s-br), 7.09-7.05 (4H, m), 6.87-6.80 (4H,m), 3.54 (1H, m), 2.92-2.84 (2H, m), 2.69-2.62 (2H, m), 1.80-1.71 (4H,m). ¹³C NMR (75 MHz, CDCl₃) δ 154.11, 130.84, 127.91, 127.77, 121.14,116.05, 69.28, 37.89, 25.87.

Example 6 Preparation of EF21, EF22

The compounds of this series were all synthesized by the followingprocedure: PCC (107 mg, 0.50 mmol) was added in one portion at roomtemperature to a stirred solution of alcohol (103 mg. 0.33 mmol) inCH₂Cl₂ (5 mL). The reaction was stirred at room temperature for 14 hrs(TLC-analysis, no sm left), filtered over CELITE and concentrated. Thecrude product was purified by chromatography on silica gel using 20%EtOAc/hexanes. A description of each compound obtained by the aboveprocess is given below.

1,5-Bis(2,4-difluorophenyl)-pentan-3-on (EF21): white solid (92%). ¹HNMR (400 MHz, CDCl₃) δ 7.16-7.09 (2H, m), 6.80-6.72 (4H, m), 2.87 (4H,t, J=7.6 Hz), 2.69 (4H, t, J=7.6 Hz). ¹³C NMR (100 MHz, CDCl₃) δ 208.08,163.00 (dd, J=48 Hz, J=11.7 Hz), 159.73 (dd, J=49 Hz, J=12 Hz), 131.39,123.54 (d, J=15.9), 111.17 (dd, J=20.8 Hz, J=3.8 Hz), 103.84 (t, J=25.5Hz), 42.93, 23.08.

1,5-Bis(3,4-difluorophenyl)-pentan-3-on (EF22): white solid (86%). ¹HNMR (400 MHz, CDCl₃) δ 7.07-7.00 (2H, ddd), 6.98-6.92 (2H, m), 6.87-6.80(2H, m), 2.84 (4H, t, J=7.2 Hz), 2.68 (4H, t, J=7.2 Hz).

Example 7 Preparation of EF7

EF7 was obtained in a three-step synthesis. To a solution of2,5-bis(2-hydroxybenzylidene)acetone (800 mg. 3.00 mmol) in DMF (10 mL)was added imidazole (545 mg, 7.56 mmol) and DMAP (10 mg). The brightyellow solution was cooled to 0° C. and tbutyldiphenylchlorosilane (1.75mL, 6.73 mmol) was added dropwise. After stirring for 30 minutes thecooling bath was removed, and the reaction proceeded at room temperatureuntil no more starting material or monoprotected alcohol were detectableby TLC. (hexanes/EtOAc=2/1, Rf (starting material)=0.17, Rf (mono)=0.44,Rf (di)=0.78). The orange solution was poured into ice water (50 mL) andextracted with ether (3×). The combined organic layers were washed withbrine (3×), dried over MgSO₄ and concentrated. The crude product waspurified by plug chromatography on silica gel (15% EtOAc/hexanes). Theproduct (108%) was obtained as a yellow foam and contains sometbutyldiphenylsilylalcohol. It was used without further purification. ¹HNMR (400 Mhz, CDCl₃) δ 8.48 (2H, d, J=16.0 Hz), 7.75-7.71 (8H, m), 7.66(2H, dd, J=8.0 Hz, J=2.0 Hz), 7.46-7.35 (12H, m), 7.18 (2H, d, J=16.4Hz), 6.96 (2H, td), 6.89 (2H, td), 6.50 (2H, dd, J=8.0 Hz, J=1.2 Hz),1.08 (18H, s). ¹³C NMR (100 MHz, CDCl₃) δ 189.93, 154.83, 138.24,135.63, 135.38, 132.35, 130.30, 128.14, 127.92, 127.26, 125.43, 121.54,120.08, 26.81, 19.88.

Disilylprotected ketone (1.74 g, 2.34 mmol) was dissolved in THF andcooled to −78° C., where CH₃Li (1.90 mL, 2.66 mmol, 1.4 M/ether) wasadded dropwise. After stirring for 10 min the originally bright yellowsolution completely cleared, it was quenched with saturated NH₄Cl, thelayers separated and the aqueous phase extracted with ether. Thecombined organic layers were washed with brine, dried over MgSO₄ andconcentrated. The crude product was purified by chromatography on silicagel (10% EtOAc/hexanes). 60%, white foam. ¹H NMR (400 MHz, CDCl₃) δ 7.72(8H, dd, J=6.4 Hz, J=1.6 Hz), 7.53 (2H, dd, J=7.2 Hz, J=2.4 Hz), 7.41(4H, m), 7.36-7.32 (10H, m), 6.82 (4H, m), 6.45 (2H, d, J=16.4 Hz), 6.43(2H, dd, J=7.2 Hz, J=2.8 Hz), 1.67 (3H, s), 1.09 (18H, s). ¹³C NMR (100MHz, CDCl₃) δ 152.96, 135.66, 135.33, 132.81, 130.12, 128.33, 128.01,127.72, 126.42, 123.37, 121.37, 119.62, 74.02, 28.39, 26.73, 19.79.

EF7: Deprotection of the alcohol was carried out with tetrabutylammoniumfluoride (2.2 equiv) in THF. The product was obtained after columnchromatography on silica gel (30% EtOAc/hexanes) as a white solid (48%).¹H NMR (400 MHz, CDCl₃) δ 7.32 (1H, dd, J=7.6 Hz, J=1.6 Hz), 7.14-7.07(2H, m), 7.00 (1H, dd, J=8.0 Hz, J=1.6 Hz), 6.89-6.82 (3H, m), 6.75 (1H,dd, J=8 Hz, J=1.2 Hz), 6.47 (1H, d, J=9.6 Hz), 6.31 (1H, d, J=16.4 Hz),5.69 (1H, d, J=10 Hz), 5.00 (1H, s-br), 1.66 (3H, s). ¹³C NMR (100 MHz,CDCl₃) δ 153.1, 153.0, 133.9, 129.5, 129.0, 128.1, 127.8, 126.7, 123.9,123.7, 121.4, 121.2, 121.1, 116.5, 116.0, 78.0, 27.6. Anal. Calcd forC₁₈H₁₆O₂: C, 81.79; H, 6.10. Found: C, 81.61; H, 6.16. EIHRMS: m/z264.1150 (M⁺, C₁₉H₁₆O₂ requires 264.1150).

Example 8 Preparation of EF5

1,5-Bis(2-hydroxyphenyl)penta-1-en-3-ol (EF5): To a solution of1,5-bis(2-hydroxyphenyl)penta-1,4-dien-3-one (EF1) (109 mg, 0.41 mmol)in THF/methanol (10/1) (2.5 mL) was added NaBH₄ (40 mg, 1.30 mmol) inone portion at 0° C. After stirring for 30 min at this temperature thereaction was quenched with H₂O dist and cold brine, diluted with Et₂O(10 mL) and neutralized by bubbling CO₂ through the dark-red solution(color change to pale yellow). The aqueous phase was extracted withether, the combined ether layers washed with brine, dried over MgSO₄ andconcentrated. The crude product was purified by chromatography on silicagel using 30% EtOAc/hexanes. The product was obtained as a white solid(66%). ¹H NMR (300 MHz, CDCl₃) δ 7.27 (2H, dd, J=8.0 Hz, J=2.4 Hz), 7.21(1H, s-br), 7.14-7.06 (3H, m), 6.90-6.83 (3H, m), 6.80-6.74 (2H, m),6.18 (1H, dd, J=20.8 Hz, J=8.8 Hz), 5.99 (1H, s-br), 4.24 (1H,t-deformed), 3.23 (1H, s-br), 2.86 (1H, m), 2.71 (1H, m), 1.91 (2H, m).¹³C NMR (75 MHz, CDCl₃) δ 154.25, 152.96, 132.84, 130.75, 129.03,127.79, 127.62, 127.46, 125.71, 123.97, 121.12, 121.07, 116.42, 116.22,72.09, 37.57, 25.76.

Example 9 Preparation of EF6

N-(Methoxy)-1,5-bis(2-hydroxyphenyl)penta-1,4-dien-3-imine (EF6):Methoxyl-amine hydrochloride (30-35 wt % in H₂O, 0.30 mL, 1.12 mmol) wasadded in one portion to a solution of1,5-bis(2-hydroxyphenyl)penta-1,4-dien-3-one (EF1) in methanol/CHCl₃(2/3) (5 mL). The reaction proceeded at room temperature for 24 hrs,then additional methoxylamine hydrochloride (0.15 mL, 0.56 mmol) wasadded and the reaction stirred for an additional 24 hrs. Aftercompletion (TLC) the solvent was evaporated, the residue dissolved inmethanol, stirred with silica gel and purified after concentration bychromatography on silica gel using 30% EtOAc/hexanes. The product wasobtained as a pale yellow foam (86%). ¹H NMR (400 MHz, CDCl₃) δ 7.56(1H, dd, J=8.0 Hz, J=1.6 Hz), 7.48 (1H, dd, J=8.0 Hz, J=1.6 Hz), 7.41(1H, d, J=16.0 Hz), 7.39 (1H, d, J=16.8 Hz), 7.28 (1H, d, J=16.8 Hz),7.20-7.13 (2H, m), 6.97-6.90 (2H, m), 6.94 (1H, d, J=16.0 Hz), 6.80 (1H,dd, J=8.0 Hz, J=1.6 Hz), 5.53 (1H, s-br), 5.36 (1H, s-br), 4.02 (3H, s).¹³C NMR (100 MHz, CDCl₃) δ 155.43, 153.86, 153.70, 132.28, 130.25,130.10, 129.71, 128.00, 127.91, 124.16, 123.88, 123.57, 121.40, 121.30,118.45, 116.37, 116.33, 62.41.

Example 10 Preparation of MD279U and MD279L

A solution of 1,5-bis(3,4-dimethoxyphenyl)penta-1,3-dien-3-one in amixture of EtOH abs and THF (5/1) was subject to hydrogenation at 50 psiusing Raney Nickel as the catalyst for 8 hrs. Filtration through CELITEand concentration under vacuum yielded the crude product, which waspurified by chromatography on silica gel using 25% EtOAc/hexanes.

1,5-Bis(3,4-dimethoxyphenyl)penta-3-one (MD279U): 37%, white solid. ¹HNMR (400 MHz, CDCl₃) δ 6.77 (2H, d, J=8.8 Hz), 6.69 (4H, m), 3.86 (6H,s), 3.85 (6H, s), 2.84 (4H, t, J=7.6 Hz), 2.70 (4H, t, J=7.6 Hz). ¹³CNMR (100 MHz, CDCl₃) δ 209.61, 149.00, 147.50, 133.77, 120.22, 111.82,111.38, 56.07, 55.98, 45.01, 29.54. R_(f)=0.28 (EtOAc/hexanes=1/2).

1,5-Bis(3,4-dimethoxyphenyl)penta-3-ol (MD279L): 51%, white solid. ¹HNMR (400 MHz, CDCl₃) δ 6.79 (2H, d, J=8.8 Hz), 6.73 (4H, m), 3.87 (6H,s), 3.86 (6H, s), 3.68 (1H, m), 2.78-2.71 (2H, m), 2.63 (2H, ddd, J=14.0Hz, J=9.2 Hz, J=6.8 Hz), 1.79 (4H, m), 1.58 (1H, s-br). ¹³C NMR (100MHz, CDCl₃) δ 149.02, 147.34, 134.80, 120.29, 111.84, 111.39, 71.04.

Example 113,5-Bis-(α,α,α-trifluoro-2-toluoylbenzylidene)-piperidin-4-one acetate

A suspension of 1.76 g (11.49 mmol) of 4-piperidone hydrate, HCl salt,in 60.0 mL of glacial acetic acid was saturated with dry HCl gas and tothe resultant solution was added 5.0 g (28.72 mmol) ofα,α,α-trifluoro-2-tolualdehyde. The mixture was allowed to stir at roomtemperature for 72 hrs and then diluted with 50.0 mL of toluene andevaporated under vacuum. The residue was diluted twice more with 50.0 mLportions of toluene and evaporated under vacuum. The gummy residue wassuspended in 50 mL of toluene containing 5.0 mL of ethyl acetate, heatedbriefly to reflux and allowed to cool to room temperature. The solidsformed were collected by suction filtration and dried under high vacuumto afford 3.63 g (67%) of a bright yellow solid.

Example 12 3-5-Bis-(pyridinylidene)-piperidin-4-one

To a solution of 1.00 g (6.52 mmol) of 4-piperidone hydrate, HCl salt,and 1.40 g (13.05 mmol) of 2-pyridine carboxaldehyde in 91 mL of a 0.25Msolution of aqueous NaOH (22.82 mmol) was added 0.86 mL of a 25% w/wsolution (0.65 mmol) of cetyltrimethylammonium chloride. The mixture wasallowed to stir vigorously at room temperature for three hours, dilutedwith 100 ml of brine and extracted with three 50 mL portions ofmethylene chloride. The organic phase was dried over anhydrous MgSO4 andconcentrated in vacuo. The residue was recrystallized from ethyl acetateto afford 1.25 g (69%) of a red-yellow solid.

Example 133,5-Bis-(2-fluoro-3-α,α,α-trifluoromethylbenzylidene)-piperidin-4-one

To a solution of 400 mg (2.61 mmol) of 4-piperidone hydrate, HCl salt,and 1.00 g (5.21 mmol) of 2-fluoro-3-α,α,α-trifluoromethylbenzaldehydein 36 mL of a 0.25M solution of aqueous NaOH (9.11 mmol) was added 0.35mL of a 25% w/w solution (0.26 mmol) of cetyltrimethylammonium chloride.The mixture was allowed to stir vigorously at room temperature for 48hours, diluted with 100 ml of brine and extracted with two 50 mLportions of methylene chloride. The organic phase was dried overanhydrous MgSO4 and concentrated in vacuo to afford 1.02 g (86%) of ayellow foam.

Example 14

To a solution of 1.0 g (6.52 mmol) of 4-piperidone hydrate, HCl salt,and 1.88 g (13.37 mmol) of 2-chloro benzaldehyde in 95 mL of a 0.25Msolution of aqueous NaOH (22.82 mmol) was added 0.90 mL of a 25% w/waqueous solution (0.65 mmol) of cetyltrimethylammonium chloride. Themixture was allowed to stir vigorously at room temperature for 48 hours,diluted with 100 ml of brine and extracted with two 35 mL portions ofmethylene chloride. The organic phase was dried over anhydrous MgSO₄ andconcentrated in vacuo. The residue was recrystallized from ethyl acetateto give 0.92 g (41%) of a pale yellow powder.

Example 15

To a solution of 1.0 g (8.84 mmol) of 1-methylpiperidine-4-one and 3.08g (13.37 mmol) of α,α,α-trifluoro-2-tolualdehyde in 88 mL of a 0.25Msolution of aqueous NaOH (22.09 mmol) was added 1.16 mL (0.88 mmol) of a25% w/w aqueous solution of cetyltrimethylammonium chloride. The mixturewas allowed to stir vigorously at room temperature for 3 hours, dilutedwith 100 ml of brine and extracted with three 50 mL portions ofmethylene chloride. The organic phase was dried over anhydrous MgSO₄ andconcentrated in vacuo to give 3.56 g (95%) of a pale yellow powder.

Example 16 3,5-Bis-(2-pyrilidinyldene)-1-methylpiperidin-4-one

To a solution of 1.0 g (8.84 mmol) of 1-methylpiperidine-4-one and 1.89g (16.70 mmol) of 2-pyridine carboxaldehyde in 88 mL of a 0.25M solutionof aqueous NaOH (22.09 mmol) was added 1.16 mL (0.88 mmol) of a 25% w/waqueous solution of cetyltrimethylammonium chloride. The mixture wasallowed to stir vigorously at room temperature for 3 hours, diluted with100 ml of brine and extracted with three 50 mL portions of methylenechloride. The organic phase was dried over anhydrous MgSO₄ andconcentrated in vacuo to give 2.50 g (97%) of a pale yellow powder.

Example 17 3,5-Bis-(4-pyridinylidene)-1-methylpiperidin-4-one

To a solution of 1.0 g (8.84 mmol) of 1-methylpiperidine-4-one and 1.89g (16.70 mmol) of 4-pyridine carboxaldehyde in 88 mL of a 0.25M solutionof aqueous NaOH (22.09 mmol) was added 1.16 mL (0.88 mmol) of a 25% w/waqueous solution of cetyltrimethylammonium chloride. The mixture wasallowed to stir vigorously at room temperature for 3 hours, diluted with100 ml of brine and extracted with two 60 mL portions of methylenechloride. The organic phase was dried over anhydrous MgSO₄ andconcentrated in vacuo to give 2.15 g (84%) of a yellow-orange powder.

Example 18 3,5-Bis-(2,6-difluorobenzylidene)-1-methylpiperidin-4-one

To a solution of 1.0 g (8.84 mmol) of 1-methylpiperidine-4-one and 2.57g (18.09 mmol) of 2,6-difluoro benzaldehyde in 90 mL of a 0.25M solutionof aqueous NaOH (22.59 mmol) was added 1.16 mL (0.88 mmol) of a 25% w/waqueous solution of cetyltrimethylammonium chloride. The mixture wasallowed to stir vigorously at room temperature for 12 hours, dilutedwith 100 ml of brine and extracted with three 40 mL portions ofmethylene chloride. The organic phase was dried over anhydrous MgSO₄ andconcentrated in vacuo. The residue was slurried in 100 ml of boilingethyl acetate and the insolubles were removed by rapid suctionfiltration. The filtrate was concentrated in vacuo and recrystallizedfrom ethyl acetate to provide 3.01 g (94%) of a bright yellow solid.

Example 19 3,5-Bis-(2,6-difluorobenzylidene)-tropin-4-one

To a solution of 0.50 g (3.59 mmol) of tropinone and 1.05 g (7.39 mmol)of 2,6-difluoro benzaldehyde in 36 mL of a 0.25M solution of aqueousNaOH (9.04 mmol) was added 0.71 mL (0.54 mmol) of a 25% w/w aqueoussolution of cetyltrimethylammonium chloride. The mixture was allowed tostir vigorously at room temperature for 4 hours, diluted with 100 ml ofbrine and extracted with two 25 mL portions of methylene chloride. Theorganic phase was dried over anhydrous MgSO₄ and concentrated in vacuo.The residue was recrystallized from ethyl acetate to provide 840 mg(60%) of a bright yellow solid.

Example 20 3,5-Bis-(2-fluorobenzylidene)-tropin-4-one

To a solution of 0.50 g (3.59 mmol) of tropinone and 0.914 g (7.36 mmol)of 2-fluoro benzaldehyde in 36 mL of a 0.25M solution of aqueous NaOH(9.04 mmol) was added 0.47 mL (0.36 mmol) of a 25% w/w aqueous solutionof cetyltrimethylammonium chloride. The mixture was allowed to stirvigorously at room temperature for 12 hours, diluted with 100 ml ofbrine and extracted with two 50 mL portions of methylene chloride. Theorganic phase was dried over anhydrous MgSO₄ and concentrated in vacuo.The residue was recrystallized from ethyl acetate to provide

Example 21 3,5-Bis-(2-pyridinylidene)-tropin-4-one

To a solution of 0.75 g (5.39 mmol) of tropinone and 1.18 g (7.39 mmol)of 2-pyridine carboxaldehyde in 54 mL of a 0.25M solution of aqueousNaOH (13.47 mmol) was added 0.71 mL (0.54 mmol) of a 25% w/w aqueoussolution of cetyltrimethylammonium chloride. The mixture was allowed tostir vigorously at room temperature for 48 hours, diluted with 150 ml ofbrine and extracted with three 50 mL portions of methylene chloride. Theorganic phase was dried over anhydrous MgSO₄ and concentrated in vacuo.The residue was recrystallized from ethyl acetate to provide 180 mg(11%) of a tan powder.

Example 22 1,5-Bis-(2,3-dimethyoxyphenyl)-penta-1,4-dien-3-one

To a solution of 0.5 g (8.61 mmol) of acetone and 2.86 g (17.22 mmol) of2,3-dimethoxy benzaldehyde in 86 mL of a 0.25M solution of aqueous NaOH(21.59 mmol) was added 2.83 mL (2.15 mmol) of a 25% w/w aqueous solutionof cetyltrimethylammonium chloride. The mixture was allowed to stirvigorously at room temperature for 72 hours, diluted with 100 ml ofbrine and extracted with three 50 mL portions of methylene chloride. Theorganic phase was dried over anhydrous MgSO₄ and concentrated in vacuo.The residue was recrystallized from ethyl acetate to provide 1.99 g(65%) of a bright yellow solid.

Example 23 1,5-Bis-(2,3-methylenedioxyphenyl)-penta-1,4-dien-3-one

To a solution of 0.19 g (3.27 mmol) of acetone and 1.00 g (6.66 mmol) of2,3-methylenedioxy benzaldehyde in 35 mL of a 0.25M solution of aqueousNaOH (8.79 mmol) was added 0.65 mL (0.49 mmol) of a 25% w/w aqueoussolution of cetyltrimethylammonium chloride. The mixture was allowed tostir vigorously at room temperature for 2 hours, at which point it wasdiluted with 15 mL of 95% ethanol, and stirring was continued for anadditional 2 hours. The solution was saturated with sodium chloride andextracted with two 35 mL portions of methylene chloride. The organicphase was dried over anhydrous MgSO₄ and concentrated in vacuo. Theresidue was recrystallized from ethyl acetate to provide 1.03 g (98%) ofa yellow solid.

Example 24 1,5-Bis-(4-dimethylaminophenyl)-penta-1,4-dien-3-one

To a solution of 1.0 g (17.22 mmol) of acetone and 5.26 g (35.30 mmol)of 4-dimethylamino benzaldehyde in 172 mL of a 0.25M solution of aqueousNaOH (43.05 mmol) was added 2.26 mL (1.72 mmol) of a 25% w/w aqueoussolution of cetyltrimethylammonium chloride. The mixture was allowed tostir vigorously at room temperature for 72 hours, diluted with 100 ml ofbrine and extracted with two 75 mL portions of methylene chloride. Theorganic phase was dried over anhydrous MgSO₄ and concentrated in vacuo.The residue was recrystallized from ethyl acetate to provide 1.21 g(22%) of a dark red powder.

Example 25 1,5-Bis-(2,6-dimethoxyphenyl)-penta-1,4-dien-3-one

To a solution of 0.25 g (4.31 mmol) of acetone and 1.47 g (8.85 mmol) of2,6-dimethoxy benzaldehyde in 43 mL of a 0.25M solution of aqueous NaOH(10.80 mmol) was added 0.57 mL (0.43 mmol) of a 25% w/w aqueous solutionof cetyltrimethylammonium chloride. The mixture was allowed to stirvigorously at room temperature for 72 hours, diluted with 100 ml ofbrine and extracted with two 75 mL portions of methylene chloride. Theorganic phase was dried over anhydrous MgSO₄ and concentrated in vacuo.The residue was recrystallized from ethyl acetate to provide 1.10 g(72%) of a yellow powder.

Example 26 1,5-Bis-(2,3-difluorophenyl)-penta-1,4-dien-3-one

To a solution of 0.5 g (8.61 mmol) of acetone and 2.51 g (17.65 mmol) of2,3-difluoro benzaldehyde in 86 mL of a 0.25M solution of aqueous NaOH(21.52 mmol) was added 1.13 mL (0.86 mmol) of a 25% w/w aqueous solutionof cetyltrimethylammonium chloride. The mixture was allowed to stirvigorously at room temperature for 48 hours, at which point it wasdiluted with 100 ml of brine and extracted with two 75 mL portions ofmethylene chloride. The organic phase was dried over anhydrous MgSO₄ andconcentrated in vacuo. The residue was recrystallized from ethyl acetateto provide 110 mg (4%) of a yellow powder.

Example 27 (E)-3-(2-fluorobenzylidenyl)indolin-2-one

To a solution containing 2.0 g (15.02 mmol) of 2-oxindole and 2.05 g(16.52 mmol) of 2-fluoro benzaldehyde in 30 mL of abs. ethanol was added190 mg (2.25 mmol) piperidine and the mixture was refluxed for 12 hrs.The mixture was allowed to cool to room temperature and the solidsformed were collected by suction filtration and washed with two 25 mLportions of cold abs. ethanol. The recovered material was dried underhigh vacuum for 12 hrs. to afford 3.22 g (90%) of a bright yellowpowder.

Example 28 (E)-3-(2-pyridinylidenyl)indolin-2-one

To a solution containing 2.0 g (15.02 mmol) of 2-oxindole and 1.77 g(16.52 mmol) of 2-pyridine carboxaldehyde in 30 mL of abs. Ethanol wasadded 192 mg (2.25 mmol) piperidine and the mixture was refluxed for 12hrs. The mixture was allowed to cool to room temperature and the solidsformed were collected by suction filtration and washed with two 25 mLportions of cold abs. ethanol. The recovered material was dried underhigh vacuum for 12 hrs to afford 2.82 g (84%) of a pale red powder.

Example 29 (E)-3-(2,3-difluorobenzylidenyl)indolin-2-one

To a solution containing 2.0 g (15.02 mmol) of 2-oxindole and 2.20 g(16.52 mmol) of 2,3-difluoro benzaldehyde in 30 mL of abs. ethanol wasadded 192 mg (2.25 mmol) piperidine and the mixture was refluxed for 12hrs. The mixture was allowed to cool to room temperature and the solidsformed were collected by suction filtration and washed with two 25 mLportions of cold abs. ethanol. The recovered material was dried underhigh vacuum for 12 hrs.

Example 30 1,3-Bis-(2-fluorobenzylidene)indan-2-one

A solution of 1.88 g (15.13 mmol) of 2-fluorobenzaldehyde in 3.0 mL ofabs. ethanol was added over a period of 5 min to a solution containing1.00 g (7.57 mmol) of 2-indanone and 90 mg (2.27 mmol) of NaOH in 40 mLof a 1:1 mixture of abs.ethanol and water at room temperature. Themixture was allowed to stir for 12 hrs and solids formed were collectedby suction filtration and washed with cold ethanol and dried under highvacuum.

Example 31 3,5-Bis-(2-fluorobenzylidene)-piperidin-4-one-acetate EF24

4-Piperidone hydrochloride monohydrate (307 mg, 2.00 mmol) was suspendedin glacial acetic acid (8 mL) and saturated with HCl gas at roomtemperature. To the resulting clear solution 2-fluorobenzaldehyde (0.59mL, 5.60 mmol) was added and the reaction allowed to stand at roomtemperature for 48 h. The forming yellow crystals were filtered off,washed with EtOH abs and dried under vacuum. Further purification wasnot necessary.

Yellow crystals (91%). ¹H NMR (400 MHz, d₆-DMSO) δ 10.11 (1H, s-br),7.90 (2H, s), 7.57 (2H, qd, J=7.6 Hz, J=1.6 Hz), 7.51 (2H, td, J=8.0 Hz,J=1.2 Hz), 7.37 (4H, q, J=10.0 Hz), 4.37 (4H, s), 3.60-3.20 (1H, s-br),1.91 (3H, s). ¹³C NMR (100 MHz, d₆-DMSO) δ 181.95, 172.04, 160.33 (d,J=249 Hz), 132.57 (d, J=9 Hz), 131.81 (d, J=4 Hz), 131.05, 129.86,124.96 (d, J=3 Hz), 121.49 (d, J=13 Hz), 116.1 (d, J=21 Hz), 43.79,21.11. HREIMS: m/z 311.1123 (M⁺-HOAc, C₁₉H₁₅NOF₂ requires 311.1122).

Example 32 Cell Viability and VEGF/TF Inhibition Analysis

As described above, the effect of the compounds of the present inventionon Neutral Red uptake, VEGF production, and TF production was measuredfor a variety of human cancer cell lines. Some of this data is alsosummarized in FIGS. 1-5. Note that, as applied to compounds EF4, MD6 andMD10 in the figures, the term “known” means that the listed compoundshave appeared in the literature, but without any suggestion that thosecompounds exhibit anti-angiogenic properties or usefulness as a cancertreatment. Table 1 below lists the results for selected curcumin analogsof the present invention in comparison to the results for curcumin andother known chemotherapeutic and anti-angiogenic agents for RPMI-7951cells.

TABLE 1 Characteristics of Selected Novel Curcumin Analogs in the HumanMelanoma Cell Line RPMI-7951 as Measured by the Neutral Red, VEGFELISA^(a) and TF ELISA^(d) Assays; Comparison with Melanoma andAnti-Angiogenesis Agents Neutral Red VEGF^(c) TF^(c) Uptake, %^(b) ELISAELISA Compound 5 μM 20 μM 5 μM 20 μM 5 μM 20 μM DMSO (0.1%) 100 37137054 Curcumin 100 14 3989 714 7974 1026 Series I MD6 97 6 2826 273 1753ND MD10 82 6 1585 315 1793 ND EF-1 92 8 1923 234 ND ND EF-2 46 7 1295 841009 ND EF-3 77 8 2092 230 ND ND EF-4 98 4 2159 199 ND ND EF-8 97 8 2208319 ND ND EF-9 88 11 1868 705 ND ND EF-10 ND 8 ND 257 ND ND Series IIMD279L 100 100 2235 1603 5791 5858 MD279U 100 100 2511 779 7200 5316EF-15 100 100 2361 894 7506 7663 Melanoma Chemotherapeutic AgentsDecabazine 100 100 1136 1531 ND ND Cisplatin 100 100 KnownAnti-angiogenic Agent Thalidomide 100 100 1810 2827 ND ND^(a)Measurement of the extent of suppression of the vascular endothelialgrowth factor (VEGF) vs. DMSO control and curcumin. ^(b)% of control.^(c)In concentration units of pg/ml/well. ^(d)Measurement of the extentof suppression of tissue factor (TF) vs. DMSO control and curcumin.

As indicated in the table and accompanying figures, two series ofanalogs were discovered. The Series I analogs proved to inhibit VEGFproduction and simultaneously inhibit cell growth for several cancercell lines. Some of the compounds within this group were also moreefficacious than TAXOL in preventing growth of a human breast cancercell line and more potent than curcumin and CISPLATIN in inhibitingproliferation of normal human and transformed murine VECs.

The Series II analogs, which include EF15, EF19-22, MD279L and MD279U,selectively blocked VEGF production without causing cell death. Thesecompounds were also not cytotoxic to normal or malignant VECs.

These results indicate that the analogs of the present invention candirectly inhibit tumor and vascular endothelial cell growth as well asshut down the production of VEGF which is vital for tumor-inducedangiogenesis. Thus, the results suggest that the novel Series I analogsare potential anti-cancer/anti-angiogenic agents, while the Series IIcompounds are promising anti-angiogenic drugs with little toxicity tonormal VECs.

Example 33

TABLE 2 Vascular Endothelial Growth Factor (VEGF) Production by HumanMelanoma Cell Line, RPMI-7951 measured by VEGF ELISA assay CellViability VEGF Concentrations 5 μM 20 μM 5 μM 20 μM of compounds (%) (%)(pg/ml) (%) (pg/ml) (%) DMSO (0.1%) 100 100 4197 100 4197 100 Curcumin105 47 4111 98 221 5 Our synthetic curcumin analogs which inhibit VEGFproduction and cell growth. EF-1 98 43 2573 61 0 0 EF-2 68 42 1522 36 00 EF-3 88 43 2827 67 0 0 EF-4 102 38 2920 70 0 0 EF-5 n.d. 84 n.d. n.d.n.d. 41 EF-6 n.d. 72 n.d. n.d. n.d. 45 EF-7 n.d. 78 n.d. n.d. n.d. 27EF-8 94 54 2276 54 35 1 EF-9 92 70 1925 46 595 14 EF-10 n.d. 54 n.d.n.d. 217 5 EF-11 n.d. 93 n.d. n.d. n.d. 51 EF-12 n.d. 92 n.d. n.d. n.d.70 EF-13 n.d. 92 n.d. n.d. n.d. 53 EF-14 n.d. 86 n.d. n.d. n.d. 36 EF-2512 n.d.  216* 21 n.d. n.d. A231L n.d. 77 n.d. n.d. n.d. 25 A231U n.d. 80n.d. n.d. n.d. 34 A232 n.d. 78 n.d. n.d. n.d. 40 A239 n.d. 86 n.d. n.d.n.d. 40 Our synthetic curcumin analogs which inhibit VEGF production,but not cell growth. EF-15 112 111 1187 28 938 22 A279L 110 109  904 220 0 A279U 112 113 1197 28 1179 18 Drugs Currently in Market**Decarbazine 91 99 1171 28 1293 31 ***Thalidomide 105 97 1865 44 238757 Other Tested Compounds MD6 101 42 3200 76 30 1 MD10 91 42 1866 44 0 0BA 3 93 102 4860 116 2035 48 BA 4 90 84 4708 112 3348 80 Values are amean of the duplicate assays. *DMSO (0.1%): VEGF 1045 pg/ml = 100%**Decarbazine: Chemotherapeutic drug currently used for treatment ofhuman melanoma. ***Thalidomide: Anti-angiogenic drug currently underclinical trial. n.d.: not done

Example 34

TABLE 3 Vascular Endothelial Growth Factor (VEGF) and Tissue Factor (TF)Production by Human Prostate Cancer Cell lines, DU-145 and PC-3 measuredby VEGF ELISA and TF ELISA, respectively All compounds are used at 20 μMand DMSO (solvent control), 0.1% at a final concentration. VEGF TFDU-145 PC-3 DU-145 PC-3 pg/ml pg/ml pg/ml pg/ml DMSO 18760 ± 2633 2103 ±100 7699 ± 406 203 ± 6 (100%) (100%)  (100%)  (100%)  Curcumin 17957 ±1910 2088 ± 103 7134 ± 301  138 ± 10  (96%) (99%) (93%) (68%) Oursynthetic curcumin analogs A 26565 ± 9818 1729 ± 59  n.d. n.d. (142%)(82%) B 15321 ± 5607 2168 ± 279 n.d. n.d.  (82%) (99%) C 20559 ± 92241734 ± 397 n.d. n.d. (110%) (82%) EF-1 19616 ± 4624 325 ± 75 12879 ±149  107 ± 9 (105%) (15%) (167%) (53%) EF-2 7516 ± 1915  26 ± 44 5540 ±364  73 ± 10  (40%)  (1%) (72%) (36%) EF-3 25901 ± 620  917 ± 261 6687 ±188 140 ± 8 (138%) (44%) (87%) (69%) EF-4 10274 ± 4467  48 ± 83 5180 ±420  33 ± 7  (55%)  (2%) (67%) (16%) A231U 27875 ± 4446  984 ± 349 n.d.n.d. (149%) (47%) A271a 24190 ± 2160 1196 ± 438 n.d. n.d. (129%) (57%)MD6 18428 ± 3377 1820 ± 283 n.d. n.d.  (98%) (87%) MD10  7830 ± 2262 611 ± 248 5886 ± 332  0 ± 0  (42%) (29%) (76%)  (0%) BA-8 40883 ± 66391648 ± 229 n.d. n.d. (218%) (78%) Values are a mean of the triplicateassays and S.D. n.d.: not done As you can see, DU-145 has a 20-foldhigher level of VEGF production than PC-3 cells. This level is seen onlyone other cell line, MDA-MB-231 breast cancer cell. Greater the VEGF andTF production, higher concentration of compounds will be required toinhibit the same percentage, e.g., effect of E-2 on DU-145 and PC-3cells. Both cell lines do not produce basic FGF (bFGF) at all.

Example 35

TABLE 4 Vascular Endothelial Growth Factor (VEGF) Production by HumanProstate Cancer Cell Lines, DU-145 & PC-3 measured by VEGF ELISA assayAll compounds are used at 20 μM and DMSO (solvent control), 0.1% at afinal concentration. DU-145 VEGF PC-3 VEGF (pg/ml) (%) pg/ml) (%) DMSO18760 ± 2633 100 2103 ± 100 100 Curcumin 17957 ± 1910 96 2088 ± 103 99Our synthetic curcumin analogs A 26565 ± 9818 142 1729 ± 59  82 B 15321± 5607 82 2168 ± 279 103 C 20559 ± 9224 110 1734 ± 397 82 EF-1 19616 ±4624 105 325 ± 75 15 EF-2  7516 ± 1915 40  26 ± 44 1 EF-3 25901 ± 620 149  917 ± 261 44 EF-4 10274 ± 4467 55  48 ± 83 2 EF-5 20140 ± 2874 1072748 ± 416 131 A231L 27927 ± 4466 149 1687 ± 250 80 A231U 27875 ± 4446149  984 ± 349 47 A232 25308 ± 4722 135 1955 ± 437 93 A239 27229 ± 2148145 1678 ± 293 80 A271a 24190 ± 2160 129 1196 ± 438 57 A272 24155 ± 4635129 1864 ± 339 89 A277a 27403 ± 3143 146 2007 ± 30  95 A277b 27124 ±1346 145 1868 ± 38  89 MD6 18428 ± 3377 98 1820 ± 283 87 MD10  7830 ±2262 42  611 ± 248 29 BA-8 40883 ± 6639 218 1648 ± 229 78 As you cansee, DU-145 has a 20-fold higher level of VEGF production than PC-3cells. This level is seen only one other cell line, MDA-MB-231 breastcancer cell. Greater the VEGF production, higher concentration ofcompounds will be required to inhibit the same percentage, e.g., effectof E-2 on DU-145 and PC-3 cells. Both cell lines do not produce basicFGF at all. Values are a mean of the triplicate assays and S.D.

Example 36

TABLE 5 Tissue Factor (TF) Production by Human Melanoma Cell Line,RPMI-7951 measured by TF ELISA assay TF Concentrations 1 μM 5 μM 20 μMof compounds (pg/ml) (%) (pg/ml) (%) (pg/ml) (%) DMSO (0.1%) 7054 1007054 100 7054 100 Curcumin n.d. 8871 126 1026 15 Our synthetic curcuminanalogs EF-2 n.d. 5780 82 1009 14 EF-15 n.d. 7506 106 7663 109 EF-32n.d. n.d. 8654 123 A231U n.d. 5473 78 5981 85 A279L n.d. 5791 82 5858 83A279U n.d. 7200 102 5316 75 MD6 6400 91 1753 25 n.d. MD10 7966 113 179325 n.d. BA-1 n.d. n.d. 6669 95 BA-3 n.d. n.d. 2088 30 BA-4 n.d. 7043 1002120 30 n.d.: not done. Values are a mean of the duplicate assays.

Example 37

TABLE 6 Tissue Factor (TF) Production by Human Breast Cancer Cell Line,MDA-MB-231 measured by TF ELISA assay TF Concentrations 0.5 μM 5 μM 10μM 20 μM of compounds (pg/ml) (%) (pg/ml) (%) (pg/ml) (%) (pg/ml) (%)DMSO (0.1%) 45753 100 45753 100 45753 100 45753 100 Curcumin n.d. 45814100 44727 98 n.d. Our synthetic curcumin analogs EF-2 37697 82 n.d. n.d.n.d. A279L n.d. n.d. n.d. 42601 93 A279L n.d. n.d. n.d. 42190 92 EF-15n.d. n.d. n.d. 46394 101 n.d.: not done. Values are a mean of theduplicate assays.

Example 38

A number of the compounds of the present invention were screened foractivity using the NCI Anti-Tumor Screen. The results are given inTables 7-9 below. Sixty human tumor cell lines were treated for 48 hourswith 10-fold dilutions of compounds at a minimum of five concentrations(0.01 μM-100 μM). Sulforhodamine B (SRB) assay was used to calculatecell viability or growth. GI50 refers to the concentration at which thedrug inhibits tumor cell growth by 50%. LC50 refers to the concentrationat which the drug causes 50% tumor cell death. EF 24 and EF 25 representthe average of three separate experiments.

TABLE 7 Median Growth Inhibitory Concentration (GI50, μM) of Compoundsin NCI Anti-Tumor Screen COMPOUNDS Panel/Cell Line EF4 EF7 EF9 EF11 EF15EF19 EF24 EF25 Curcumin CISPLATIN Leukemia CCRF-CEM 1.4 12.5 3.1 22.041.5 18.2 0.2 0.3 3.2 0.2 HL-60 (TB) 1.9 17.7 4.2 21.3 49.9 18.1 0.4 1.17.9 0.1 K-562 2.5 14.9 3.5 27.4 78.5 19.0 0.3 0.6 3.2 1.0 MOLT-4 2.2 7.79.3 19.7 >100 2.6 0.5 0.4 5.0 0.3 RPMI-8226 0.6 14.8 2.4 18.2 49.8 21.70.2 0.2 2.0 0.5 SR 1.6 15.2 1.2 21.5 52.0 22.7 0.1 0.2 3.2 0.1 Non-SmallCell Lung Cancer A549 2.6 14.9 8.9 38.7 >100 18.0 1.9 2.5 12.6 0.8 EKVX2.3 17.3 11.8 30.6 64.0 17.5 0.9 1.4 15.8 1.6 HOP-62 2.9 16.3 16.683.3 >100 18.0 0.7 1.5 4.0 0.3 HOP-92 2.8 12.7 16.3 23.5 >100 17.5 3.02.4 ND 0.6 NCI-H226 19.0 18.7 24.3 >100 >100 19.9 3.6 2.2 20.0 0.8NCI-H23 1.8 17.6 12.3 35.8 >100 22.8 0.9 1.7 5.0 0.1 NCI-H322M 2.7 16.916.7 36.5 >100 20.3 1.4 1.9 15.8 1.3 NCI-H460 2.3 17.6 12.7 30.7 >10017.9 81.0 1.2 7.9 0.1 NCI-H522 2.9 16.2 13.4 33.3 69.9 18.2 0.9 0.9 5.00.8 Colon Cancer COLO 205 2.0 17.5 7.6 39.6 >100 17.5 0.8 2.0 12.6 4.0HCC-2998 1.7 18.4 3.3 20.5 31.6 17.7 1.1 1.5 3.2 0.1 HCT-116 2.2 17.61.9 36.8 >100 17.8 0.2 0.8 3.2 1.3 HCT-15 2.2 17.2 4.9 42.6 49.1 24.80.2 0.7 4.0 1.6 HT29 2.2 17.8 4.1 45.9 46.1 17.9 0.3 2.0 5.0 1.3 KM121.6 17.1 2.3 19.1 55.7 17.6 0.2 0.3 5.0 2.0 SW-620 2.7 16.2 3.2 32.579.8 17.7 0.2 0.5 4.0 0.8 CNS Cancer SF-268 3.0 19.8 13.7 24.6 >100 21.50.6 1.4 6.3 0.2 SF-295 3.4 15.5 14.2 53.8 80.9 18.7 85.0 1.7 7.9 0.3SF-539 2.0 17.3 2.6 39.9 >100 16.9 0.3 0.5 3.2 0.4 SNB-19 3.2 17.5 14.748.0 >100 16.1 0.7 2.2 7.9 1.0 SNB-75 2.0 11.8 11.2 20.3 98.8 19.0 2.01.8 6.3 0.6 U251 2.0 16.1 2.1 35.7 95.1 17.4 0.2 0.7 5.0 0.4 MelanomaLOX IMVI 2.2 17.2 3.4 40.3 >100 20.0 0.2 0.2 2.5 0.3 MALME-3M 2.3 17.517.8 40.7 >100 18.0 2.3 1.9 12.6 0.2 M14 1.9 15.7 4.1 33.9 >100 18.3 1.21.8 4.0 0.3 SK-MEL-2 2.4 16.8 14.5 24.0 31.7 18.4 2.6 2.0 15.8 1.3SK-MEL-28 2.9 17.4 18.6 42.5 83.7 17.1 2.1 2.4 5.0 0.8 SK-MEL-5 1.6 15.512.1 23.2 53.9 17.1 1.3 1.5 7.9 0.5 UACC-257 2.9 16.9 16.0 29.8 55.417.9 1.8 1.6 12.6 1.0 UACC-62 1.5 12.2 13.9 21.3 66.7 15.1 1.6 1.7 6.30.3 Ovarian Cancer IGROV1 2.9 15.9 3.8 35.9 >100 17.2 0.8 0.8 7.9 0.6OVCAR-3 3.7 15.6 14.1 41.8 >100 17.9 1.1 0.9 6.3 1.0 OVCAR-4 2.5 14.115.2 28.7 >100 17.4 0.7 2.0 10.0 0.3 OVCAR-5 2.6 16.7 20.0 89.2 >10018.5 1.3 1.7 15.8 1.3 OVCAR-8 2.9 17.0 3.4 47.1 >100 18.0 0.4 0.8 7.91.0 SK-OV-3 6.7 16.6 15.4 >100 >100 18.5 1.7 2.4 7.9 1.3 Renal Cancer786-0 2.3 16.4 3.5 41.3 >100 17.0 0.2 0.4 3.2 0.3 A498 2.2 17.2 2.4 34.223.3 16.3 0.9 1.3 15.8 2.0 ACHN 3.3 15.7 16.5 41.0 >100 17.8 1.1 1.512.6 0.5 CAKI-1 3.1 14.3 15.0 29.2 >100 21.8 0.8 1.8 12.6 0.3 RXF 3930.5 11.0 2.3 39.0 23.5 18.8 0.6 0.6 3.2 0.8 SN12C 2.3 15.2 17.134.2 >100 17.4 1.0 1.4 7.9 1.0 TK-10 3.0 17.4 17.9 41.2 >100 17.4 3.12.2 15.8 1.3 UO-31 1.6 13.5 9.6 28.4 96.8 18.2 0.6 1.2 12.6 0.8 ProstateCancer PC-3 2.1 17.2 4.8 34.9 >100 17.5 0.3 0.5 7.9 1.6 DU-145 1.4 16.42.1 29.9 >100 18.1 0.7 1.2 15.8 0.4 Breast Cancer MCF-7 2.7 18.0 3.028.7 44.6 17.7 0.2 0.3 3.2 0.4 NCI/ADR-RES 2.7 19.3 30.7 86.1 >100 25.30.9 1.6 7.9 0.8 MDA-MB-231 3.5 19.8 14.1 25.6 >100 22.4 1.3 1.9 20.0 3.2HS 578T 5.1 22.4 9.9 52.1 >100 21.9 1.0 5.1 10.0 1.3 MDA-MB-435 1.8 17.53.2 31.4 61.2 17.1 0.6 1.0 3.2 1.6 MDA-N 1.6 16.1 2.9 36.9 67.8 16.9 0.51.0 2.5 0.8 BT-549 2.4 17.1 6.5 32.9 >100 17.9 0.7 1.5 5.0 1.3 T-47D 1.77.7 4.4 24.7 18.5 15.5 1.3 1.3 7.9 2.0 Mean 2.4 15.8 7.2 34.7 75.9 17.80.7 1.1 6.7 0.7 ND—Not Determined

As shown, EF4, EF24 and EF 25 exhibited a lower GI50 than thechemotherapeutic agent CISPLATIN for several cell types.

TABLE 8 Median Lethal Concentration (LC50, μM) of Compounds in NCIAnti-Tumor Screen COMPOUNDS Panel/Cell Line EF4 EF7 EF9 EF19 EF24 EF25Curcumin CISPLATIN Leukemia CCRF-CEM 59.1 79.5 >100 72.3 70.9 >100 <10039.8 HL-60 (TB) 53.5 76.0 >100 66.8 >100 >100 <100 50.1^(#) K-562 91.470.7 >100 90.1 75.4 91.8 <100 50.1 MOLT-4 69.5 64.7 >100 75.6 66.2 >10079.4 50.1 RPMI-8226 >100 92.7 95.6 >100 54.9 70.9 <100 50.1 SR >100 76.877.9 82.6 68.2 76.3 79.4 50.1 Non-Small Cell Lung Cancer A549 >100 53.371.0 65.8 89.4 71.8 79.4 50.1 EKVX 37.2 56.6 49.1 57.7 9.4 24.2 79.450.1 HOP-62 69.1 63.1 60.7 58.7 28.8 37.2 63.1 50.1 HOP-92 41.4 54.970.4 59.3 23.3 35.4 ND 50.1 NCI-H226 >100 78.8 >100 75.7 48.3 58.0 <10050.1 NCI-H23 9.4 62.9 62.3 96.8 5.3 12.9 <100 50.1 NCI-H322M 42.3 56.055.6 59.2 17.0 32.2 63.1 50.1 NCI-H460 40.2 66.8 60.9 57.5 50.1 38.963.1 39.8 NCI-H522 >100 >100 57.1 75.4 22.9 14.1 79.4 50.1 Colon CancerCOLO 205 8.7 55.9 52.8 55.9 8.0 33.4 63.1 50.1 HCT-2998 5.9 56.8 32.256.1 5.0 5.3 31.6 19.9 HCT-116 13.2 57.6 7.7 56.3 1.8 11.1 50.1 50.1HCT-15 >100 68.9 64.7 >100 13.6 16.1 79.4 50.1 HT29 29.1 60.6 41.9 62.657.3 67.5 <100 50.1 KM12 6.5 55.5 14.6 56.1 3.4 10.7 63.1 50.1 SW-62076.3 65.9 62.6 63.7 4.4 75.3 79.4 50.1 CNS Cancer SF-268 53.2 >100 >10085.3 55.6 43.3 <100 50.1 SF-295 79.5 55.1 58.8 69.7 20.6 35.0 50.1 50.1SF-539 24.7 55.7 23.1 55.3 7.6 16.8 31.6 50.1 SNB-19 42.3 55.9 58.8 57.319.3 37.3 63.1 50.1 SNB-75 21.5 49.0 48.8 57.5 8.8 14.0 50.1 50.1 U25118.5 54.4 12.9 55.8 0.8 5.8 50.1 39.8 Melanoma LOX IMVI >100 73.4 67.674.4 6.7 3.6 <100 50.1 MALME-3M 24.3 68.8 73.4 59.6 25.2 19.3 50.1 39.8M14 13.2 54.0 48.6 68.7 27.7 19.4 50.1 50.1 SK-MEL-2 25.3 57.0 58.9 57.019.5 12.6 79.4 50.1 SK-MEL-28 33.8 62.9 61.1 55.6 33.1 6.6 50.1 50.1SK-MEL-5 5.7 53.7 53.1 55.5 6.4 6.6 50.1 7.9 UACC-257 57.8 55.2 62.156.3 13.5 8.2 50.1 50.1 UACC-62 6.1 49.6 52.8 53.2 4.5 14.4 50.1 15.8Ovarian Cancer IGROV1 43.9 60.9 46.2 55.6 7.1 14.5 79.4 50.1 OVCAR-340.7 54.5 52.1 59.3 16.2 23.5 63.1 50.1 OVCAR-4 18.4 53.1 61.6 65.1 17.223.3 <100 39.8 OVCAR-5 55.9 58.4 63.3 61.1 9.2 8.4 79.4 50.1OVCAR-8 >100 86.4 85.3 67.3 31.3 23.0 79.4 50.1 SK-OV-3 63.6 56.2 53.765.9 12.8 26.9 50.1 50.1 Renal Cancer 786-O 15.9 54.8 49.6 55.4 1.6 32.639.8 50.1 A498 16.2 55.6 23.0 54.7 3.4 6.0 63.1 50.1 ACHN 32.7 54.0 54.856.3 5.6 18.2 63.1 50.1 CAKI-1 >100 52.3 93.4 67.8 27.2 53.7 50.1 50.1RXF 393 30.8 49.2 17.9 58.4 3.6 4.6 50.1 50.1 SN12C 21.5 54.4 62.3 55.95.4 12.0 63.1 50.1 TK-10 22.6 55.9 56.5 56.3 27.8 24.6 63.1 50.1 UO-316.2 51.3 49.5 56.6 4.5 68.1 50.1 25.1 Prostate Cancer PC-3 28.6 55.641.0 56.7 7.2 24.8 63.1 50.1 DU-145 7.0 54.8 29.0 56.6 5.4 20.1 63.150.1 Breast Cancer MCF-7 44.1 65.9 42.2 57.1 5.0 44.7 <100 50.1NCI/ADR-RES >100 >100 >100 >100 60.6 71.0 <100 50.1 MDA-MB-231 >100 74.657.7 66.3 9.1 17.2 <100 50.1 HS 578T >100 >100 >100 >100 81.4 72.3 <10050.1 MDA-MB-435 8.0 56.0 35.9 55.6 7.4 16.3 39.8 39.8 MDA-N 20.2 56.933.2 55.2 5.6 20.0 31.6 39.8 BT-549 26.1 56.8 47.3 64.7 4.7 13.6 39.850.1 T-47D 50.8 55.4 56.1 68.9 51.9 92.9 <100 50.1 Mean 33.9 61.7 52.564.6 13.2 24.2 66.0 46.0

EF4, EF9, EF24, and EF25 exhibited a lower LC50 than thechemotherapeutic agent CISPLATIN for several cell types.

Human tumor and endothelial cell lines were treated for 72 hours withcompounds at a minimum of four concentrations between 0.1 μM-40 μM.Neutral Red assay was used to calculate cell viability. The numbers inTable 9 are representative of at least three separate experiments.

TABLE 9 Median Growth Inhibitory Concentration (GI50, μM) of Compoundsin Emory Laboratory Cell Screen COMPOUNDS Panel/Cell Line EF2 EF4 EF25EF34 MD6 MD10 MD283 MD286 MD287 Curcumin Melanoma RPMI 7951 0.8 3.6 0.6ND 1.9 2.2 1.4 1.0 0.7 6.3 Breast Cancer MDA-MB-231 1.5 ND 0.8 0.8 ND ND1.8 ND ND 11.6 MDA-MB-435 3.3 ND 1.8 ND ND ND 1.9 ND ND 16.3 HUVECS 1.7ND 1.5 ND 3.8 14.0 3.1 2.3 6.8 25 Mean 1.8 3.6 1.2 0.8 2.9 8.1 2.1 1.73.8 14.8

Many modifications and other embodiments of the invention will come tomind to one skilled in the art to which this invention pertains havingthe benefit of the teachings presented in the foregoing descriptions andthe associated drawings. Therefore, it is to be understood that theinvention is not to be limited to the specific embodiments disclosed andthat modifications and other embodiments are intended to be includedwithin the scope of the appended claims. Although specific terms areemployed herein, they are used in a generic and descriptive sense onlyand not for purposes of limitation.

1. A pharmaceutical formulation comprising a compound of the formula:

wherein: the dashed lines indicate the presence of optional doublebonds; X is O, S, or NR₅, R₁ and R₂ are independently selected from thegroup consisting of halogen, hydroxyl, alkoxy, CF₃, alkyl, substitutedalkyl, alkenyl, alkynyl, cycloalkyl, substituted cycloalkyl, aryl,substituted aryl, alkaryl, arylalkyl, heteroaryl, substitutedheteroaryl, heterocycle, substituted heterocycle, amino, alkylamino,dialkylamino, carboxylic acid, carboxylic ester, carboxamide, nitro,cyano, azide, alkylcarbonyl, acyl, and trialkylammonium; R₃ and R₄ areindependently selected from the group consisting of H, halogen,hydroxyl, alkoxy, CF₃, alkyl, substituted alkyl, alkenyl, alkynyl,cycloalkyl, substituted cycloalkyl, aryl, substituted aryl, alkaryl,arylalkyl, heteroaryl, substituted heteroaryl, heterocycle, substitutedheterocycle, amino, alkylamino, dialkylamino, carboxylic acid,carboxylic ester, carboxamide, nitro, cyano, azide, alkylcarbonyl, acyl,and trialkylammonium; R₅ is selected from the group consisting of H,alkyl, substituted alkyl, acyl, alkoxycarbonyl, aminocarbonyl,alkylaminocarbonyl, and dialkylaminocarbonyl; or a pharmaceuticallyacceptable salt thereof; and a pharmaceutically acceptable carrier. 2.The pharmaceutical formulation of claim 1, wherein X is NR₅.
 3. Thepharmaceutical formulation of claim 2, wherein R₅ is a H.
 4. Thepharmaceutical formulation of claim 2, wherein R₁ and R₂ areindependently selected from the group consisting of halogen, hydroxyl,and alkoxy.
 5. The pharmaceutical formulation of claim 2, wherein R₁ andR₂ are hydroxyl.
 6. The pharmaceutical formulation of claim 2, whereinR₁ and R₂ are independently selected from the group of halogen.
 7. Thepharmaceutical formulation of claim 2, wherein R₁ and R₂ are F.
 8. Thepharmaceutical formulation of claim 1, wherein X is O.
 9. Thepharmaceutical formulation of claim 8, wherein R₁ and R₂ areindependently selected from the group consisting of halogen, hydroxyl,and alkoxy.
 10. The pharmaceutical formulation of claim 1, wherein X isS.
 11. The pharmaceutical formulation of claim 10, wherein R₁ and R₂ areindependently selected from the group consisting of halogen, hydroxyl,and alkoxy.
 12. The pharmaceutical formulation of claim 1, wherein R₁and R₂ are independently selected from the group consisting of halogen,hydroxyl, and alkoxy.
 13. A method of treating cancerous tissue in asubject, comprising administering to the subject an effective amount ofa compound of the formula

wherein: the dashed lines indicate the presence of optional doublebonds; X is O, S, or NR₅, R₁ and R₂ are independently selected from thegroup consisting of halogen, hydroxyl, alkoxy, CF₃, alkyl, substitutedalkyl, alkenyl, alkynyl, cycloalkyl, substituted cycloalkyl, aryl,substituted aryl, alkaryl, arylalkyl, heteroaryl, substitutedheteroaryl, heterocycle, substituted heterocycle, amino, alkylamino,dialkylamino, carboxylic acid, carboxylic ester, carboxamide, nitro,cyano, azide, alkylcarbonyl, acyl, and trialkylammonium; R₃ and R₄ areindependently selected from the group consisting of H, halogen,hydroxyl, alkoxy, CF₃, alkyl, substituted alkyl, alkenyl, alkynyl,cycloalkyl, substituted cycloalkyl, aryl, substituted aryl, alkaryl,arylalkyl, heteroaryl, substituted heteroaryl, heterocycle, substitutedheterocycle, amino, alkylamino, dialkylamino, carboxylic acid,carboxylic ester, carboxamide, nitro, cyano, azide, alkylcarbonyl, acyl,and trialkylammonium; and R₅ is selected from the group consisting of H,alkyl, substituted alkyl, acyl, alkoxycarbonyl, aminocarbonyl,alkylaminocarbonyl, and dialkylaminocarbonyl; or a pharmaceuticallyacceptable salt thereof; wherein said cancerous tissue is selected fromthe group consisting of breast cancer, colon cancer, prostate cancer,skin cancer, leukemia, non-small cell lung cancer, CNS cancer, ovariancancer, and renal cancer.
 14. The method of claim 13, wherein theeffective amount comprises an amount sufficient to inhibit VEGFproduction in the cancerous tissue.
 15. The method of claim 13, whereinthe effective amount comprises an amount sufficient to inhibit TFproduction in the cancerous tissue.
 16. The method of claim 13, whereinsaid administering step comprises administering an effective amount ofthe compound in a pharmaceutically acceptable carrier.
 17. The method ofclaim 13, wherein X is NR₅.
 18. The method of claim 17, wherein R₅ is aH.
 19. The method of claim 17, wherein R₁ and R₂ are independentlyselected from the group consisting of halogen, hydroxyl, and alkoxy. 20.The method of claim 17, wherein R₁ and R₂ are hydroxyl.
 21. The methodof claim 17, wherein R₁ and R₂ are independently selected from the groupof halogen.
 22. The method of claim 17, wherein R₁ and R₂ are F.
 23. Themethod of claim 13, wherein X is O.
 24. The method of claim 23, whereinR₁ and R₂ are independently selected from the group consisting ofhalogen, hydroxyl, and alkoxy.
 25. The method of claim 13, wherein X isS.
 26. The method of claim 25, wherein R₁ and R₂ are independentlyselected from the group consisting of halogen, hydroxyl, and alkoxy. 27.The method of claim 13, wherein R₁ and R₂ are independently selectedfrom the group consisting of halogen, hydroxyl, and alkoxy.
 28. A methodof treating an inflammatory skin condition in a subject, comprisingadministering to the subject an effective amount of a compound of theformula

wherein: the dashed lines indicate the presence of optional doublebonds; X is O, S, or NR₅, R₁ and R₂ are independently selected from thegroup consisting of halogen, hydroxyl, alkoxy, CF₃, alkyl, substitutedalkyl, alkenyl, alkynyl, cycloalkyl, substituted cycloalkyl, aryl,substituted aryl, alkaryl, arylalkyl, heteroaryl, substitutedheteroaryl, heterocycle, substituted heterocycle, amino, alkylamino,dialkylamino, carboxylic acid, carboxylic ester, carboxamide, nitro,cyano, azide, alkylcarbonyl, acyl, and trialkylammonium; R₃ and R₄ areindependently selected from the group consisting of H, halogen,hydroxyl, alkoxy, CF₃, alkyl, substituted alkyl, alkenyl, alkynyl,cycloalkyl, substituted cycloalkyl, aryl, substituted aryl, alkaryl,arylalkyl, heteroaryl, substituted heteroaryl, heterocycle, substitutedheterocycle, amino, alkylamino, dialkylamino, carboxylic acid,carboxylic ester, carboxamide, nitro, cyano, azide, alkylcarbonyl, acyl,and trialkylammonium; and R₅ is selected from the group consisting of H,alkyl, substituted alkyl, acyl, alkoxycarbonyl, aminocarbonyl,alkylaminocarbonyl, and dialkylaminocarbonyl; or a pharmaceuticallyacceptable salt thereof.
 29. The method of claim 28, wherein theinflammatory skin condition is dermatitis.
 30. The method of claim 28,wherein the inflammatory skin condition is related to skin cancer.